Methods of treating ttp with immunoglobulin single variable domains and uses thereof

ABSTRACT

The present invention is based on the finding that administration of polypeptides comprising at least one Immunoglobulin single variable domains against vWF to human TTP patients provides a significant decrease in the time to response. The invention provides a polypeptide comprising at least one immunoglobulin single variable domain (ISVD) against von Willebrand Factor (vWF) for use in treating a vWF-related disease in a human in need thereof. The invention further relates to dosage unit forms, kits and medical uses for treating TTP.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/319,467, filed Dec. 16, 2016, which is a national stage filing under35 U.S.C. § 371 of International Application No. PCT/EP2015/063493,filed Jun. 16, 2015, which was published under PCT Article 21(2) inEnglish, and claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application Ser. No. 62/030,817, filed Jul. 30, 2014, theentire contents of each of which is incorporated by reference herein inits entirety.

1. FIELD OF THE INVENTION

The present invention is based on the finding that administration ofpolypeptides comprising at least one Immunoglobulin single variabledomain against vWF to human TTP patients provides a significant decreasein the time to response and less complications. The invention provides apolypeptide comprising at least one immunoglobulin single variabledomain (ISVD) against von Willebrand Factor (vWF) for use in treating avWF-related disease in a human in need thereof. The invention furtherrelates to dosage unit forms, kits and medical uses for treating TTP.

2. BACKGROUND OF THE INVENTION 2.1 Role of vWF in Platelet Aggregation

The multimeric plasma protein von Willebrand Factor (vWF) is essentialfor recruiting circulating platelets to the damaged vessel wall uponvascular injury. This recruitment is mediated through binding of the vWFA1-domain with the platelet receptor glycoprotein GPIb-IX-V.

Upon expression by endothelial cells, vWF is secreted into thecirculation as ultra-large multimers or ultra-large vWF (ULvWF). Thesemultimers are processed into smaller regular sized multimers throughenzymatic cleavage by ADAMTS13. In these regular sized multimers of vWF,the GPIb-IX-V platelet receptor binding site in the A1 domain is crypticand will not spontaneously react with platelets. A conformationalactivation of the GPIb-IX-V platelet receptor binding site in the A1domain is triggered by immobilisation or under conditions of shearstress resulting in platelet adhesion and subsequently in thrombusformation.

2.2 Role of vWF and vWF Processing in Pathophysiology of TTP

Thrombotic thrombocytopenic purpura (“TTP”) is a rare andlife-threatening disease of the blood coagulation system, in whichaccumulation of ULvWF multimers has been implicated, leading to anincreased risk of thrombus formation in small blood vessels due toexcessive platelet aggregation. The condition is characterised bysystemic platelet aggregation in the microcirculation, producingfluctuating ischaemia in many organs. If sustained, this may causetissue infarction, associated with profound thrombocytopenia anderythrocyte fragmentation.

ULvWF multimers have the natural ability to spontaneously interact withthe platelet receptor GPIb-IX-V. In healthy subjects, these ULvWFmultimers are immediately processed into regular sized vWF multimers viacleavage by the vWF protease ADAMTS13. However, ADAMTS13 activity wasfound to be severely deficient in hereditary TTP as well as acquiredidiopathic TTP. The majority of patients with TTP have autoantibodiesagainst ADAMTS13 resulting in impaired processing of the ULvWFmultimers. As a consequence, the A1 domain of the ULvWF isconstitutively active and readily interacting with the GPIb-IX-Vplatelet receptor. This eventually results in formation of thecharacteristic blood clots found in the TTP patient population.

The current therapy of TTP with Plasma Exchange (abbreviated herein as“PE” or “PEX”) and transfusion provides replacement ADAMTS13 and removesantibodies against the enzyme, thus progressively leading to anormalisation of ULvWF processing. However, this treatment requiresmultiple exchanges and transfusions over many days, during which timethere is no direct pharmacological targeting of the active process ofULvWF-mediated platelet aggregation.

Although the introduction of PE and transfusion has significantlyreduced the mortality rates from TTP over the last three decades, thecondition still carries a significant risk of mortality and morbidity.The mortality rate of acute bouts in acute idiopathic TTP, in patientsmanaged with the current therapies remains in the order of 10% to 30%(Vesely et al. Blood 2003; 102: 60-68; Allford et al. Br. J. Haematol.2003; 120: 556-573; Sadler et al. Hematology. Am. Soc. Hematol. Educ.Program. 2004; 407-423). In the case of secondary TTP, PE andtransfusion are recognised to be less effective and the mortality rateis considerably higher. In the cases when the disease is secondary topregnancy, in which PE is regarded as reasonably effective the mortalityrate of an acute bout of UP is approximately 25%, rising to over 40% incases with concurrent pre-eclampsia (Martin et al. Am. J. Obstet.Gynecol. 2008; 199: 98-104). However, in cases secondary to, forexample, underlying malignancies or bone marrow transplant the mortalityrate remains at 40% to 60% despite the use of such treatment regimens(Sadler et al. 2004 supra; Elliott et al. Mayo Clin. Proc. 2003; 78:421-430; Kremer Hovinga and Meyer Curr. Opin. Hematol. 2008; 15:445-450.)

Given the continuing significant level of mortality from TTP and theobserved complications of PE and transfusion, there is a clear need forthe development of additional therapeutic approaches to supplement, orpotentially reduce the need for, these methods of treatment.

The research conducted into TTP over the past three decades has improvedthe understanding of the pathophysiology of the disease allowing for thepotential development of novel agents targeting the underlying diseaseprocesses. Nevertheless, there are no currently approved therapies forTTP, and although there are newer therapies currently undergoingevaluation, the studies of these potential treatments are at arelatively early stage.

Immunoglobulin single variable domains (ISVDs) against vWF have beendescribed in, for example WO2004/015425, WO2004/062551, WO2006/074947,WO2006/122825, WO2009/115614 and WO2011/067160.

It has been shown that ISVDs against vWF (e.g. ALX 0081) avidly bind tomultimeric vWF, thereby blocking the interaction of any sizes andactivation stages of multimeric vWF with the GPIb-IX-V plateletreceptor. The interaction of ALX 0081 with vWF is highly specific and itdoes not interact with human blood cells or platelets. Furthermore, itsinterference with the platelet GPIb-IX-V receptor is selective throughthe binding of the vWF A1 domain and it does not affect the capacity ofvWF to interact with fibrillar collagens or with collagen type VI. Ithas also been shown that ISVDs against vWF (e.g. ALX 0081) do not affectthe activity of the (remaining) vWF-protease ADAMTS13, nor do theyinterfere with the binding of FVIII to vWF.

In a phase I study it has been shown that ALX 0081 is safe and welltolerated in healthy volunteers.

However, the human healthy volunteers are not predictive for theefficacy of ISVDs against vWF in general or ALX 0081 specifically in theunderlying pathology of TTP patients, vWF is abnormal in quantity aswell as quality in TTP patients. Although it is accepted that ULvWF doesnot function normally in hemostasis in TTP patients, the underlyingmechanism is not understood. In TTP patients, higher vWF levels areexpected during acute episodes (Lotta et al. 2011 J Thromb Haemost 9:1744-51; Stufano et al. 2012 J Thromb Haemost 10:728-730).

Due to the lack of a relevant animal model, no in vivo efficacy of ALX0081 to neutralise ULvWF has been demonstrated.

Therefore, it remains to be elucidated whether polypeptides comprisingat least one ISVD against vWF, such as ALX 0081, are beneficial in TTPpatients, whether polypeptides comprising at least one ISVD against vWF,such as ALX 0081, have a positive effect over PE, and what an effectivetreatment and dose regimen would be.

There is a need for improved therapies for TTP patients.

3. SUMMARY OF THE INVENTION

The present invention is based on the unexpected finding that theadministration of polypeptides comprising at least one ISVD against vWFto human TTP patients provides a decrease of 2 days in thetime-to-response, objectified by a recovery of platelets 150,000/pi.Platelet count increase is a sign of diminished pathological plateletaggregation, thereby decreasing the thrombotic process initiated by theplatelet-vWF complexes characteristic of this disease. The Hazard Ratio(“HR”) of placebo over the polypeptide of the invention was anastonishing of 2.2 with 95% CI (1.28, 3.78), p=0.013. This response wasconfirmed up to 48 hours after the time-to-response. Hence, proof ofconcept of the polypeptide of the invention was demonstrated withstatistically significant and clinically meaningful reduction of time toconfirmed platelet response. Furthermore, there was a reduction in thenumber of exacerbations from 11 in the Placebo arm to 3 in the treatmentarm. There were no deaths in the treatment arm compared to 2 deaths inthe Placebo arm.

Moreover, the present clinical study with TTP patients also demonstratesthat the polypeptides of the present invention (e.g. ALX 0081) are welltolerated agents and, in particular, that the potential for the risk ofbleeding appears to be present but low and manageable. The currentlyavailable data demonstrate, therefore, that the reduction in PE andtransfusion and their associated complications are achieved withoutsignificant adverse effects from the use of the polypeptides of theinvention itself. This represents a clear safety benefit for the use ofthe polypeptides of the invention in the treatment of patients with TTP.

Hence, the administration of polypeptides comprising at least one ISVDagainst vWF to human TTP patients provides an unexpectedly decreasedtime-to-response, a sustained and prolonged effect, reducedexacerbations, reduced hospitalization, reduced morbidity, reduceddeaths and/or a reduced number of PEs.

The current therapy of TTP with PE and transfusion provides replacementADAMTS13 and removes antibodies against the enzyme, thus progressivelyleading to a normalisation of ULvWF processing. However, this treatmentrequires multiple exchanges and transfusions over many days, duringwhich time there is no direct pharmacological targeting of the activeprocess of ULvWF-mediated platelet aggregation.

It has now furthermore unexpectedly been shown that the polypeptides ofthe present invention do not interfere with the enzyme replaced byplasma transfusion. It has been demonstrated that the polypeptides ofthe invention (e.g. ALX 0081) can be utilized, in combination with PEand transfusion, to directly inhibit the continuing formation of smallthrombi and platelet consumption in the microvasculature. This permitsmore rapid control of the underlying thrombotic process and accompanyingplatelet consumption, with the benefits of a reduced degree of ischaemicand haemorrhagic complications. It also results in a more rapid clinicalrecovery and less morbidity with a shorter period and reduced number ofPEs and transfusions. Indeed, an analysis on the specific and clinicallyrelevant organ damage biomarkers LDH, troponin T or I and creatininesuggested that more rapidly curtailing microvascular tissue ischemiacould be expected to have a clinical benefit. In addition, thedemonstrated inhibition of ULvWF-mediated platelet interaction by thepolypeptides of the invention (e.g. ALX 0081) and the observedantithrombotic effects raise the potential for its longer-term use afterpatients have recovered from an acute bout of TTP to prevent relapses ofthe disease. A reduced frequency of acute bouts of TTP represents asignificant benefit, with a potential for a reduction in the mortalityand morbidity associated with TTP and a further reduction in the needfor PE and transfusions over a patient's lifetime.

While a more rapid recovery from TTP and a reduction in exacerbationsand relapses is a clear clinical benefit in terms of treatment efficacy,the reduction in the duration and frequency of PE and transfusion alsoprovides additional benefits in terms of patient safety. Although PE andtransfusion are currently regarded as the standard treatment in themanagement of TTP (Scully et al. Br. J. Haem. 2012; 158:323-335), theprocedures carry the risk of significant complications. The PE procedurerequires high fluid volumes and flow rates necessitating the use ofcentral venous dual lumen haemodialysis catheters. Complications fromthe procedure include haemorrhage from catheter insertion, sepsis,catheter thrombosis, pneumothorax, fluid overload, hypoxia andhypotension (Fontana et al. Semin. Hematol. 2004; 41: 48-59; George J.Intensive Care Med. 2007; 22: 82-91; Howard et al. Transfusion 2006; 46:154-156; Rizvi et al. Transfusion 2000; 40: 896-901; Nguyen et al.Transfusion 2009; 49: 392-394). Anaphylactoid reactions complicate 0.25%to 0.5% of procedures (Allford et al 2003 supra; George 2007 supra). Inaddition, the infusion of plasma containing blood products can cause anon-infective TRALI. This condition is recognized as one of the mostfrequent causes of transfusion-related fatalities with an incidenceestimated to be 0.02% to 0.05% per plasma containing unit with a dailyaverage of 17 plasma units, the daily risk can be calculated to a rangeof 0.34% to 0.85%. Most patients with TTP require multiple PEs andtransfusions. Patients with acute idiopathic TTP require dailytreatments, and an average of approximately 16 treatments is required toachieve remission (Allford et al. 2003 supra). In refractory cases thefrequency of treatment may be increased to twice-daily (Allford et al.2003 supra). In the case of patients with familial TTP, regularprophylactic plasma infusions at two to three week intervals arerecommended (Lammle et al. J. Thromb. Haemost. 2005; 3: 1663-1675).Anaphylaxis and TRALI thus represent clear risks to patients with TTPwhose treatment requires such a frequency and regularity of PEs andtransfusions. While it is thought that this risk may be lower ifsolvent/detergent (S/D) treated plasma is used instead of fresh frozenplasma, the use of large volumes of S/D plasma may be associated with anincreased risk of venous thromboembolism (Allford et al. 2003 supra;Fontana et al. 2004 supra). Overall, it is estimated that approximately30% to 40% of patients will experience adverse effects from PE andtransfusion, and the mortality rate from the procedure is of the orderof 2% to 3% (George et al. Semin. Hematol. 2004; 41: 60-67; George 2007supra). Hence, the reduction in the duration and frequency of PE andtransfusion also provides additional benefits in terms of patientsafety.

Following recovery from a bout of TTP, many patients describe cognitiveabnormalities for many years and report troublesome problems withmemory, concentration, decreased energy and fatigue. Such symptoms havea negative impact on the quality of patients' daily lives. Furthermore,this deficit in quality of life may occur in all patients who have TTP,regardless of the aetiology and severity (Lewis et al. Transfusion 2009;49: 118-124). It is thought that these symptoms may be reflective of theresidual effects of tissue ischaemia. On this basis, it can bereasonably proposed that a more rapid recovery from TTP and thelimitation of thrombus formation in the microvasculature that thepolypeptides of the present invention, such as ALX 0081, provide,results in an improved longer-term outcome for the patients in terms oftheir quality of life.

Accordingly, the present invention provides methods for treating oralleviating vWF-related diseases in a subject by administering to thesubject a polypeptide comprising at least one ISVD against vWF, whereinthe amount of the polypeptide administered is effective to reduce thetime-to-response, to reduce exacerbations, to reduce hospitalization, toreduce ischemia, to reduce the death toll and/or to reduce the number ofrequired PEs. The present invention provides specific dose ranges anddosing schedules for the polypeptides of the invention that result inone or more of these effects on vWF-related disease. In particular, theinvention provides pharmacologically active agents, compositions,methods and/or dosing schedules that have certain advantages compared tothe agents, compositions, methods and/or dosing schedules that arecurrently used and/or known in the art, including the requirement toless frequently give PE. These advantages will become clear from thefurther description below.

Accordingly, the present invention provides a polypeptide comprising atleast one immunoglobulin single variable domain (ISVD) against vonWillebrand Factor (vWF) for use in treating a vWF-related disease in ahuman in need thereof, comprising administering to said human a firstdose of 1-80 mg, such as 5-40 mg, preferably 10 mg of said polypeptide.

The present invention provides a polypeptide as described herein,wherein said administering said polypeptide is followed within 5 min to8 h by performing a first Plasma Exchange (PE).

The present invention provides a polypeptide as described herein,wherein said administering of said first dose is preceded by performinga preceded Plasma Exchange (PE), preferably within 36 h, such as within32 h, 30 h, 28 h, 26 h, 24 h, 22 h, 20 h, 18 h, 16 h, 14 h, 12 h, 10 h,8 h, for instance within 7 h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30min, 20 min, 15 min, 10 min or even 5 min of said first PE.

The present invention provides a polypeptide as described herein,wherein said first PE is followed by administering a second dose of 1-80mg, such as 5-40 mg, preferably 10 mg, of said polypeptide, preferablyby subcutaneous injection, preferably within 1-60 min, more preferablywithin 30 min of said first PE.

The present invention provides a polypeptide as described herein,wherein said preceded PE is performed within 36 h, preferably 32, 30,28, 26, 24, 22, 20, 18, or 16 h, preferably about 24 h of said first PE.

The present invention provides a polypeptide as described herein,wherein said polypeptide is administered parenterally, preferably bysubcutaneous, intraperitoneal, intravenous or intramuscular injection,preferably by an intravenous (i.v.) bolus push injection.

The present invention provides a polypeptide as described herein,wherein said administering said polypeptide is followed by performing aPE within 5 min to 8 h, such as within 10 min to 6 h or 15 min to 4 h,for instance within 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30min, 20 min, 15 min, 10 min or even 5 min.

The present invention provides a polypeptide as described herein,wherein said treating a vWF-related disease in a human in need thereof,further comprises:

-   (i) performing a PE; and (followed by)-   (ii) administering a dose of 1-80 mg, such as 5-40 mg of said    polypeptide 5 min to 4 h after said PE of step (i); and-   (iii) optionally measuring the platelet count and/or ADAMTS13    activity of said patient,    wherein step (i) and step (ii) are repeated once per day, preferably    until the platelet count of said patient is ≥150000/μl and/or said    ADAMTS13 activity is at least 10% such as at least 15%, 20%, 25%,    30%, 35%, 45% or even 50% of the ADAMTS13 reference activity.

The present invention provides a polypeptide as described herein,further comprising administering once per day a dose of 1-80 mg, such as5-40 mg, preferably 10 mg of said polypeptide for at least 5, 10, 15,20, 25, 30, 40, 50 60, 90 or even 120 days after the platelet count ofsaid patient is ≥150.000/μl for the first time.

The present invention provides a polypeptide as described herein,further comprising administering once per day a dose of 1-80 mg, such as5-40 mg, preferably 10 mg of said polypeptide until said human entersremission.

The present invention provides a polypeptide as described herein,comprising administering said polypeptide until the ADAMTS13 activity isat least 10% such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50%of the ADAMTS13 reference activity.

The present invention provides a polypeptide as described herein,wherein said dose is about 1-80 mg, or 5-40 mg, such as 1, 2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40,50, 60, 70 or 80 mg, preferably about 10 mg of said polypeptide.

The present invention provides a polypeptide as described herein,wherein said human suffers from an acute episode of TTP, an exacerbationof TTP or a relapse of TTP.

In a preferred aspect, the present invention provides a polypeptidecomprising at least one immunoglobulin single variable domain (ISVD)against von Willebrand Factor (vWF) for use in treating a vWF-relateddisease in a human in need thereof, comprising

-   (1) optionally performing a preceded Plasma Exchange (PE);-   (2) administering to said human a first dose of 1-80 mg, such as    5-40 mg, preferably 10 mg of said polypeptide, and if step (1) is    performed preferably within 36 h, such as within 32 h, 30 h, 28 h,    26 h, 24 h, 22 h, 20 h, 18 h, 16 h, 14 h, 12 h, 10 h, 8 h, for    instance within 7 h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30 min,    20 min, 15 min, 10 min or even 5 min of (the end of) step (1);-   (3) performing a Plasma Exchange (PE), optionally within 5 min to 8    h, such as within 10 min to 6 h or 15 min to 4 h, for instance    within 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30 min, 20    min, 15 min, 10 min or even 5 min of step (2);-   (4) administering a further dose of 1-80 mg, such as 5-40 mg,    preferably 10 mg of said polypeptide preferably within 5 min to 8 h,    such as within 10 min to 6 h or 15 min to 4 h, for instance within 8    h, 7 h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30 min, 20 min, 15    min, 10 min or even 5 min of (the end of) step (3);-   (5) repeating step (3) and step (4) once per day; optionally until    the platelet count of said patient is ≥150000/μl and/or said    ADAMTS13 activity is at least 10% such as at least 15%, 20%, 25%,    30%, 35%, 45% or even 50% of the ADAMTS13 reference activity.-   (6) optionally administering once per day a dose of 1-80 mg, such as    5-40 mg, preferably 10 mg of said polypeptide for at least 5, 10,    15, 20, 25, 30, 40, 50 60, 90 or even 120 days after the platelet    count of said patient is ≥150.000/μl for the first time or until the    ADAMTS13 activity is at least 10% such as at least 15%, 20%, 25%,    30%, 35%, 45% or even 50% of the ADAMTS13 reference activity.

In addition, the present invention provides a polypeptide comprising twoanti-human vWF ISVDs for use in preventing (the symptoms of) a relapseof an vWF-related disease in a human, by administering to the human 1-80mg, such as 5-40 mg, preferably 10 mg doses of said polypeptide.

The present invention provides a polypeptide as described herein,wherein said ISVD against vWF comprises at least one immunoglobulinsingle variable domain binding to SEQ ID NO: 20.

The present invention provides a polypeptide as described herein,wherein said ISVD against vWF comprises a heavy chain variable domainwhich is derived from a conventional four-chain antibody or a heavychain variable domain which is derived from a heavy chain antibody or aNanobody.

The present invention provides a polypeptide as described herein,wherein said Nanobody is a VHH.

The present invention provides a polypeptide as described herein,wherein said the ISVD against vWF essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

a) CDR1 comprises or essentially consists of:

-   -   the amino acid sequence YNPMG; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence YNPMG;        and        b) CDR2 comprises or essentially consists of:    -   the amino acid sequence AISRTGGSTYYPDSVEG; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AISRTGGSTYYPDSVEG; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AISRTGGSTYYPDSVEG;        and        c) CDR3 comprises or essentially consists of:    -   the amino acid sequence AGVRAEDGRVRTLPSEYTF; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AGVRAEDGRVRTLPSEYTF; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AGVRAEDGRVRTLPSEYTF.

The present invention provides a polypeptide as described herein, inwhich:

-   -   a) CDR1 is YNPMG (SEQ ID NO: 20);    -   b) CDR2 is AISRTGGSTYYPDSVEG (SEQ ID NO: 21); and    -   c) CDR3 is AGVRAEDGRVRTLPSEYTF (SEQ ID NO: 22).

The present invention provides a polypeptide as described herein,wherein the ISVD against vWF is represented by SEQ ID NO: 19 (12A02H1).

The present invention provides a polypeptide as described herein,comprising or consisting of at least two ISVDs against vWF.

The present invention provides a polypeptide as described herein,wherein each ISVD of said at least two ISVDs against vWF essentiallyconsists of 4 framework regions (FR1 to FR4, respectively) and 3complementarity determining regions (CDR1 to CDR3, respectively), inwhich:

a) CDR1 comprises or essentially consists of:

-   -   the amino acid sequence YNPMG; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence YNPMG;        and        b) CDR2 comprises or essentially consists of:    -   the amino acid sequence AISRTGGSTYYPDSVEG; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AISRTGGSTYYPDSVEG; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AISRTGGSTYYPDSVEG;        and        c) CDR3 comprises or essentially consists of:    -   the amino acid sequence AGVRAEDGRVRTLPSEYTF; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AGVRAEDGRVRTLPSEYTF; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AGVRAEDGRVRTLPSEYTF.

The present invention provides a polypeptide as described herein, inwhich each ISVD against vWF essentially consists of 4 framework regions(FR1 to FR4, respectively) and 3 complementarity determining regions(CDR1 to CDR3, respectively), in which:

a) CDR1 is YNPMG (SEQ ID NO: 20); b) CDR2 is AISRTGGSTYYPDSVEG (SEQ IDNO: 21); and c) CDR3 is AGVRAEDGRVRTLPSEYTF (SEQ ID NO: 22),

The present invention provides a polypeptide as described herein,wherein said polypeptide comprises or consists of SEQ ID NO:s 1-19,preferably SEQ ID NO: 19.

The present invention provides a polypeptide as described herein,wherein said ISVD against vWF is a single chain polypeptide comprisingone or more immunoglobulin single variable domains.

The present invention provides a polypeptide as described herein,wherein said ISVD against vWF is monovalent or multivalent.

The present invention provides a polypeptide as described herein,wherein said ISVD against vWF is monospecific or multispecific.

The present invention provides a polypeptide as described herein,wherein one or more immunoglobulin single variable domains areCDR-grafted, humanized, camelized, de-immunized, or selected by phagedisplay.

The present invention provides a polypeptide as described herein,wherein said ISVD against vWF comprises an amino acid sequence which isat least 90% identical to SEQ ID NO: 1.

The present invention provides a polypeptide as described herein,comprising two anti-human vWF immunoglobulin single variable domains(ISVDs) and an anti-human serum albumin (HSA) ISVD

The present invention provides a polypeptide as described herein,wherein said polypeptide is formulated in a pharmaceutically acceptableformulation.

The present invention provides a polypeptide as described herein,wherein said formulation comprises a citrate or phosphate buffer with apH in the range of 5.0 to 7.5.

The present invention provides a polypeptide as described herein,wherein said formulation is suitable for parenteral administration, suchas one or more selected from intravenous injection, subcutaneousinjection, intramuscular injection or intraperitoneal injection.

The present invention provides a polypeptide as described herein,wherein said formulation is in liquid, lyophilized, spray-dried,reconstituted lyophilized or frozen form.

The present invention provides a kit or an article of manufacture,comprising a container containing the polypeptide as described herein orthe formulation as described herein, and instructions for use.

The present invention provides a kit or article of manufacture asdescribed herein, wherein the formulation is present in a vial or aninjectable syringe.

The present invention provides a kit or article of manufacture asdescribed herein, wherein the formulation is present in a prefilledinjectable syringe.

The present invention provides a kit or article of manufacture asdescribed herein, wherein the syringe or a vial is composed of glass,plastic, or a polymeric material chosen from a cyclic olefin polymer orcopolymer.

The present invention provides a formulation comprising:

-   (a) a vWF binder at a concentration from about 0.1 mg/mL to about 80    mg/mL;-   (b) an excipient chosen from sucrose, glycine, mannitol, trehalose    or NaCl at a concentration of about 1% to about 15% (w/v);-   (c) Tween-80 at a concentration of about 0.001% to 0.5% (v/v); and-   (d) a buffer chosen from citrate buffer at a concentration of about    5 mM to about 200 mM such that the pH of the formulation is about    6.0 to 7.0 and a phosphate buffer at a concentration of about 10 mM    to about 50 mM such that the pH of the formulation is about 6.5 to    7.5,    for use in treating a vWF-related disease in a human in need    thereof, by administering to the human a 1-80 mg, such as 5-40 mg    dose, preferably 10 mg of said polypeptide, wherein said dose is    followed within 5 min to 8 h, such as 15 min to 4 h by a first    Plasma Exchange (PE).

The present invention provides a pharmaceutical unit dosage formsuitable for parenteral administration to a patient, preferably a humanpatient, comprising a polypeptide as described herein or a formulationas described herein.

The present invention provides a polypeptide as described herein,wherein said vWF-related disease is chosen from acute coronary syndrome(ACS), transient cerebral ischemic attack, unstable or stable anginapectoris, stroke, myocardial infarction or thrombotic thrombocytopenicpurpura (TTP).

The present invention provides a method for the treatment of a humanpatient susceptible to or diagnosed with a disease characterized by avWF-related disease, comprising administering an effective amount of apolypeptide comprising at least one immunoglobulin single variabledomain (ISVD) against von Willebrand Factor (vWF) to the human patient.

The present invention provides a method of treating or preventing avWF-related disease, such as TTP, comprising administering to a human,1-80 mg, such as 5-40 mg, preferably 10 mg dose of a polypeptidecomprising at least one immunoglobulin single variable domain (ISVD)against von Willebrand Factor (vWF), thereby reducing one or moresymptoms associated with the vWF-related disease.

The present invention provides a treatment as described herein, whereinsaid administering a polypeptide as described herein is followed within5 min to 8 h, such as 15 min to 4 h by performing a first PlasmaExchange (PE).

The present invention provides a treatment as described herein, whereinsaid administering of a polypeptide as described herein is preceded byperforming a preceded Plasma Exchange (PE), within 36 h, preferably 32,30, 28, 26, 24, 22, 20, 18, or 16 h, preferably about 24 h of said firstPE.

The present invention provides a treatment as described herein, whereinsaid first PE is followed by administering a second dose of 1-80 mg,such as 5-40 mg, preferably 10 mg of a polypeptide as described hereinwithin 5 min to 8 h, such as within 10 min to 6 h or 15 min to 4 h, forinstance within 8 h, 7 h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30 min,20 min, 15 min, 10 min or even 5 min, for instance wherein said seconddose of said polypeptide is administered within 1-60 min, such as 30 minof said first PE, preferably by subcutaneous injection.

The present invention provides a treatment as described herein, furthercomprising:

-   (i) performing a PE; (followed by)-   (ii) administering a dose of 1-80 mg such as 5-40 mg, preferably 10    mg of a polypeptide as described herein 15 min to 4 h after said PE    of step (i); and-   (iii) optionally measuring the platelet count and/or ADAMTS13    activity of said patient, wherein step (i) and step (ii) are    repeated once per day optionally until the platelet count of said    patient is ≥150000/μl and/or the ADAMTS13 activity is at least 10%    such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50% of the    ADAMTS13 reference activity.

The present invention provides also a treatment as described herein,further comprising administering once per day a dose of 1-80 mg, such as5-40 mg, preferably 10 mg of a polypeptide as described herein for atleast 5, 10, 15, 20, 25, or even 30 days after the platelet count ofsaid patient is ≥150.000/μl.

The present invention provides a treatment as described herein, furthercomprising administering once per day a dose of 1-80 mg, such as 5-40mg, preferably 10 mg of a polypeptide as described herein until saidhuman enters remission.

The present invention provides a treatment as described herein,comprising administering said polypeptide until the ADAMTS13 activity isat least 10% such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50%of the ADAMTS13 reference activity.

In an embodiment, the present invention relates to a method for reducingthe risk of and/or preventing an acute episode of a vWF-related diseasein a human in need thereof, comprising or consisting of: (i)administering to said human a dose of 5-40 mg, preferably 10 mg, of apolypeptide comprising at least one immunoglobulin single variabledomain (ISVD) against von Willebrand Factor (vWF); whereinadministration of said polypeptide reduces the risk of and/or preventsan acute episode of a vWF-related disease. Preferably, said risk isreduced by a factor 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, suchas 3, 4, 5, 6, 7, 8, 9, or even 10, or even more such as 20, 50 or even100. Preferably, said risk is reduced by 10% or even more such as 20%,30%, 40%, 50%, 60% or more, such as 80% or even 100%.

In an embodiment, the present invention relates to a method as describedherein, wherein said step (i) of administering the polypeptide of theinvention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10times, or even more than 10 times, such as 20 times, preferably morethan 30 times or even more.

In an embodiment, the present invention relates to a method as describedherein, wherein said step (i) of administering the polypeptide of theinvention is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10days, or even more than 10 days, such as 20 days, preferably more than30 days, such as 2 months, 3 months, 4 months, 5 months, 6 months oreven more.

In an embodiment, the present invention relates to a method as describedherein, wherein said dose is administered 1 time per day or two timesper day.

In an embodiment, the present invention relates to a method as describedherein, further comprising

-   (ii) measuring the ADAMTS13 activity of said patient;-   (iii) comparing said ADAMTS13 activity with a reference ADAMTS13    activity; and-   (iv) if said ADAMTS13 activity is lower than 30%, such as 20%, 15%    or 10% of said reference ADAMTS13 activity, then repeating said    step (i) of administering the polypeptide of the invention.

In an embodiment, the present invention relates to a method as describedherein, wherein said ADAMTS13 activity of said patient is measured everyday, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at leastonce every week.

In an embodiment, the present invention relates to a method as describedherein, wherein step (i) of administering the polypeptide of theinvention is repeated until said ADAMTS13 activity is at least 10%, 15%,such 20%, or even 30% or higher of said reference ADAMTS13 activity.

In an embodiment, the present invention relates to a method as describedherein, wherein step (i) is repeated until said ADAMTS13 activity is atleast 10%, 15%, such as 20% or 30% of said reference ADAMTS13 activityon at least 2 consecutive measurements. Preferably, said 2 consecutivemeasurements are at least 24 h, more preferably 48 h apart, such as atleast 3 days apart, or even more such as, 4, 5, 6, or even 7 days apart,preferable a week apart,

In an embodiment, the present invention relates to a method as describedherein, wherein said step (i) of administering the polypeptide of theinvention is repeated for at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 days, or even more than 10 days, such as 20 days, preferably morethan 30 days or even more, after said ADAMTS13 activity is at least 10%or 15%, such as 20% or 30% of said reference activity on at least 2consecutive measurements.

In an embodiment, the present invention relates to a method as describedherein, further comprising

-   -   measuring the ADAMTS13 activity of said patient;    -   comparing said ADAMTS13 activity with a reference ADAMTS13        activity; and    -   if said ADAMTS13 activity is ≥10%, such as more than 15%, or        more than 20% or 30% of said reference ADAMTS13 activity, then        repeating said step (i) of administering the polypeptide of the        invention for at most 30 days, such as at most 20 days, or even        15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or even 1 day.

In an embodiment, the present invention relates to a method for reducingthe risk of and/or preventing an acute episode of a vWF-related diseasein a human in need thereof, comprising at least the following steps:

-   (i) measuring the ADAMTS13 activity of said patient;-   (ii) comparing said ADAMTS13 activity with a reference ADAMTS13    activity; and-   (iii) if said ADAMTS13 activity is lower than 30%, 20%, 15% or 10%    of said reference activity, then administering to said human a dose    of 5-40 mg of a polypeptide comprising at least one immunoglobulin    single variable domain (ISVD) against von Willebrand Factor (vWF);

In an embodiment, the present invention relates to a method as describedherein, wherein

-   -   the risk of organ damage, ischaemic damage and/or microthrombi        formation is reduced by 10%, 20%, 30%, preferably by at least        40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even to        100%;    -   the risk of organ damage, ischaemic damage and/or microthrombi        formation is reduced by a factor 1.2. 1.3, 1.4, 1.5, 1.75, 2 or        more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more such        as 20, 50 or even 100;    -   organ damage, ischaemic damage and/or microthrombi formation is        reduced preferably by at least 10%, 20%, 30%, 40%, or even at        least 50%, such as 60%, 70%, 80%, 90% or even to 100%;    -   organ damage, ischaemic damage and/or microthrombi formation is        reduced by a factor, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or        even 10, or even more such as 20, 50 or even 100;    -   organ damage markers, such as LDH levels, troponin T, troponin I        levels, and/or creatinine levels, return to at least 40%, or        even at least 50%, such as 60%, 70%, 80%, 90% or even to 100% of        normal levels;    -   organ damage markers, such as LDH levels, troponin T, troponin I        levels, and/or creatinine levels, improve by at least 20%, such        30% or even higher, such as 40%, or even at least 50%, such as        60%, 70%, 80%, 90% or even to 100% of normal levels. Preferably,        said organ damage, such as LDH levels, troponin T, troponin I        levels, and/or creatinine levels, markers improve in less than        30 days of treatment, preferably, in less than 20 days of        treatment, such as, less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2        days or even within 1 day.    -   the number of platelets is kept at ≥150000/μl.    -   the risk of exacerbations is reduced by at least 10%, 20%, 30%,        40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even to        100%;    -   the risk of exacerbations is reduced by a factor, 2 or more,        such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more such as        20, 50 or even 100;    -   mortality due to said vWF related disease is reduced by 10%,        20%, 30%, preferably by at least 40%, or even at least 50%, such        as 60%, 70%, 80%, 90% or even to 100%;    -   mortality due to said vWF related disease is reduced by a factor        1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more, such as 3, 4, 5,        6, 7, 8, 9, or even 10, or even more such as 20, 50 or even 100.

In an embodiment, the present invention relates to a method as describedherein, further comprising measuring the platelet number; and if saidplatelet number is lower than 150,000/μl, then repeating said step (i)of administering the polypeptide of the invention.

In an embodiment, the present invention relates to a method as describedherein, wherein said platelet number of said patient is measured everyday, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at leastevery week.

In an embodiment, the present invention relates to a method as describedherein, wherein step (i) of administering the polypeptide of theinvention is repeated until said platelet number is at least 150,000/μl.

In an embodiment, the present invention relates to a method as describedherein, wherein step (i) of administering the polypeptide of theinvention is repeated until said platelet number is at least 150,000/μlon at least 2 consecutive measurements. Preferably, said 2 consecutivemeasurements are at least 24 h, more preferably 48 h apart, such as atleast 3 days apart, or even more such as, 4, 5, 6, or even 7 days apart,preferable a week apart.

In an embodiment, the present invention relates to a method as describedherein, wherein said step (i) of administering the polypeptide of theinvention is repeated for at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 days, or even more than 10 days, such as 20 days, preferably morethan 30 days or even more, after said platelet number is at least150,000/μl on at least 2 consecutive measurements. Preferably, said 2consecutive measurements are at least 24 h, more preferably 48 h apart,such as at least 3 days apart, or even more such as, 4, 5, 6, or even 7days apart, preferably a week apart.

In an embodiment, the present invention relates to a method as describedherein, further comprising measuring the platelet number of saidpatient; and if said platelet number ≥150,000/μl, then repeating saidstep (i) of administering the polypeptide of the invention for at most30 days, such as at most 20 days, or even 15, 10, 9, 8, 7, 6, 5, 4, 3, 2days or even 1 day.

In an embodiment, the present invention relates to a method for reducingthe risk of and/or preventing an acute episode of a vWF-related diseasein a human in need thereof, comprising at least the following steps:

-   (i) measuring the platelet number of said patient; and-   (ii) if said platelet number is lower than 150,000/μl, then    administering to said human a dose of 5-40 mg of a polypeptide    comprising at least one immunoglobulin single variable domain (ISVD)    against von Willebrand Factor (vWF);    -   wherein administration of said polypeptide reduces the risk of        and/or prevents an acute episode of a vWF-related disease.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, comprising at least the following steps;

-   (i) administering to said human a first dose of 5-40 mg, preferably    10 mg of a polypeptide comprising at least one immunoglobulin single    variable domain (ISVD) against von Willebrand Factor (vWF);-   (ii) performing a first Plasma Exchange (PE), preferably within 5    min to 8 h of step (i).

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, as described herein, wherein step (i), i.e. administeringto said human the polypeptide of the invention, is preceded byperforming a preceding PE, preferably within 24 h of step (ii), i.e.performing a first PE.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, comprising at least the following steps: (i) performing aPlasma Exchange (PE); (ii) administering to said human a dose of 5-40mg, preferably 10 mg of a polypeptide comprising at least oneimmunoglobulin single variable domain (ISVD) against von WillebrandFactor (vWF). Preferably said step (i), i.e. performing a PE, and saidstep (ii) i.e. administering to said human said polypeptide of theinvention, are repeated once or twice per day, for at most for 1, 2, 3,4, 5, 6, or 7 days.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, as described herein, wherein step (ii) i.e. administeringto said human said polypeptide of the invention, is performed within 15min to 4 h of step (i), i.e. performing a PE.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, as described herein, further comprising measuring theplatelet count of said human, preferably after step (ii) i.e.administering to said human said polypeptide of the invention; and ifsaid platelet count is <150,000/μl, repeating said step (i) i.e.performing a PE, and said step (ii) i.e. administering to said humansaid polypeptide.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, as described herein, further comprising measuring theplatelet count of said human [preferably after step (ii) i.e.administering to said human said polypeptide of the invention]; andrepeating step (i), i.e. performing a PE, and step (ii) i.e.administering to said human said polypeptide, [once/twice per day] untilsaid platelet number is at least 150,000/μl on at least 2 consecutivemeasurements. Preferably, said 2 consecutive measurements are at least24 h, more preferably 48 h apart, such as at least 3 days apart, or evenmore such as, 4, 5, 6, or even 7 days apart, preferable a week apart.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, as described herein, further comprising administering onceper day a dose of 5-40 mg, preferably 10 mg of said polypeptide for atleast 1-30 days after the platelet count of said human was for the firsttime ≥150.000/μl.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, as described herein, further comprising measuring theADAMTS13 activity of said human, preferably after step (ii) i.e.administering to said human said polypeptide.

In an embodiment, the present invention relates to a method for treatingan acute episode of a vWF-related disease, such as TTP, in a human inneed thereof, as described herein, wherein step (i), i.e. performing aPE, and step (ii) i.e. administering to said human said polypeptide ofthe invention, are repeated until the ADAMTS13 activity is [for thefirst time] more than 15%, or 20% or even 30% of a reference ADAMTS13activity.

In an embodiment, the present invention relates to a method for reducingthe risk of and/or preventing ischaemic damage, organ damage and/ormicrothrombi formation [causable by a vWF-related disease] in a human inneed thereof, comprising at least the following step: (i)

-   -   administering to said human a dose of 5-40 mg/day, preferably 10        mg/day of a polypeptide comprising at least one immunoglobulin        single variable domain (ISVD) against von Willebrand Factor        (vWF); wherein administration of said polypeptide reduces the        risk of and/or prevents ischaemic damage, organ damage and/or        microthrombi formation by 10%, 20%, 30%, preferably by at least        40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even to        100%. Preferably, administration of said polypeptide reduces the        risk of and/or prevents ischaemic damage, organ damage and/or        microthrombi formation by a factor 1.2, 1.3, 1.4, 1.5, 1.6,        1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10,        or even more such as 20, 50 or even 100.

In an embodiment, the present invention relates to a method wherein saidstep (i) of administering said polypeptide is repeated for at least 1,2, 3, 4, 5, 6, 7 days, or even longer such as 1 week, 2 weeks, 3 weeks,or even longer such as 1 month or even 2 months

In an embodiment, the present invention relates to a method furthercomprising measuring ADAMTS13 activity of said patient, preferably onceper week.

In an embodiment, the present invention relates to a method wherein saidstep (i) of administering said polypeptide is repeated for at least 1,2, 3, 4, 5, 6, 7 days, or even longer such as 1 week, 2 weeks, 3 weeks,or even longer such as 1 month or even 2 months when the ADAMTS13activity is [for the first time]≥10%, such as more than 15%, or evenmore than 20% of a reference ADAMTS13 activity.

In an embodiment, the present invention relates to a method of treatinga symptom of a vWF-related disease, such as TTP, in a human sufferingfrom said disease, comprising administering to the subject a polypeptideof the invention, in an amount effective to treat the symptom of avWF-related disease in a human suffering from said disease.

In an embodiment, the present invention relates to a method ofinhibiting in a human the onset or progression of a vWF-related disease,such as TTP, the inhibition of which is effected by binding of apolypeptide comprising at least one immunoglobulin single variabledomain (ISVD) against von Willebrand Factor (vWF) to vWF, comprisingadministering to the human at a predefined interval effective inhibitorydoses of said polypeptide, wherein each administration of thepolypeptide delivers to the human from 0.1 mg per kg to 25 mg per kg ofthe human's body weight, so as to thereby inhibit the onset orprogression of the disease in the human,

In an embodiment, the present invention relates to a method of reducingthe likelihood of a human contracting ischaemic organ damage by avWF-related disease, which comprises administering to the human at apredefined dose a polypeptide comprising at least one immunoglobulinsingle variable domain (ISVD) against von Willebrand Factor (vWF),wherein each administration of the antibody delivers to the human from0.1 mg per kg to 25 mg per kg of the human's body weight, so as tothereby reduce the likelihood of the human contracting ischaemic organdamage.

4. BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Treatment flow chart.

FIG. 2 Time to first LDH normalisation curves (ITT population=subjectswith abnormal high levels at baseline).

FIG. 3 Time to Troponin T or I Normalization Curves for Subjects withAbnormal High Levels at Baseline in the Intent-To-Treat Population.

FIG. 4 Time to Creatinine Normalization Curves for Subjects withAbnormal High Levels at Baseline in the Intent-To-Treat Population.

FIG. 5: vWF levels in TTP patients: Model-predicted % decrease frombaseline of free vWF levels at the end of period 2 as a function of thedaily dose level, including patients treated with placebo. Median, 25thand 75th percentiles are indicated.

FIG. 6: PK/PD-Model of caplacizumab and free, total and complexed vWF:(A) Model-predicted caplacizumab concentration profiles after daily s.c.administration during period 1 and period 2 (with and withoutconcomitant daily PE). (B) Model-predicted free vWF levels during adaily 10 mg s.c. administration of caplacizumab and after the treatmentperiod, (C) Estimated complex caplacizumab-vWF levels during a daily 10mg s.c. administration of caplacizumab and after the treatment period.(D) Model-predicted total vWF levels during a daily 10 mg s.c.administration of caplacizumab and after the treatment period. Medianprofiles, 5th and 95th percentiles are shown.

5. DETAILED DESCRIPTION

Unless indicated otherwise, all methods, steps, techniques andmanipulations that are not specifically described in detail can beperformed and have been performed in a manner known per se, as will beclear to the skilled person. Reference is for example again made to thestandard handbooks and the general background art mentioned herein andto the further references cited therein; as well as to for example thefollowing reviews Presta, Adv. Drug Deliv. Rev. 2006, 58 (5-6): 640-56;Levin and Weiss, Mol. Biosyst. 2006, 2(1): 49-57; Irving et al., J.Immunol. Methods, 2001, 248(1-2), 31-45; Schmitz et al., Placenta, 2000,21 Suppl. A, S106-12, Gonzales et al., Tumour Biol., 2005, 26(1), 31-43,which describe techniques for protein engineering, such as affinitymaturation and other techniques for improving the specificity and otherdesired properties of proteins such as immunoglobulins.

It must be noted that as used herein, the singular forms “a”, “an”, and“the”, include plural references unless the context clearly indicatesotherwise. Thus, for example, reference to “a reagent” includes one ormore of such different reagents and reference to “the method” includesreference to equivalent steps and methods known to those of ordinaryskill in the art that could be modified or substituted for the methodsdescribed herein.

Unless otherwise indicated, the term “at least” preceding a series ofelements is to be understood to refer to every element in the series.Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the present invention.

The term “and/or” wherever used herein includes the meaning of “and”,“or” and “all or any other combination of the elements connected by saidterm”.

The term “about” or “approximately” as used herein means within 20%,preferably within 15%, more preferably within 10%, and most preferablywithin 5% of a given value or range.

Throughout this specification and the claims which follow, unless thecontext requires otherwise, the word “comprise”, and variations such as“comprises” and “comprising”, will be understood to imply the inclusionof a stated integer or step or group of integers or steps but not theexclusion of any other integer or step or group of integer or step. Whenused herein the term “comprising” can be substituted with the term“containing” or “including” or sometimes when used herein with the term“having”.

The therapeutic potential of the polypeptides of the invention, such asALX 0081, in a TTP setting was demonstrated by in vitro experimentsusing plasma from TTP patients in flow chamber experiments. In theseexperiments, endothelial cells were stimulated to produce ULvWF stringson their surface (see Example 7.2). It was demonstrated that thepolypeptides of the invention, such as ALX 0081, were able to inhibitplatelet-vWF interactions and particularly ULvWF-mediated plateletinteraction in vitro and were also shown to have no impact on ADAMTS13function. In particular, it was demonstrated that the polypeptides ofthe invention, such as ALX 0081, are able to interact with vWF in bothits active (i.e. functional for interaction with GPIb-IX-V as regularsize multimers and as ultra-large multimers) and in its inactive stage(regular size multimers prior to conformational change of A1 domain).The study demonstrated a proof of concept that the polypeptides of theinvention, such as ALX 0081, can be used to treat TTP patients. It alsoproves that the polypeptides of the invention, such as ALX 0081, do notinterfere with the ADAMTS13 activity.

The present invention is at least partly based on the finding that theadministration to human TTP patients of polypeptides comprising at leastone ISVD against vWF (also referred to herein as “polypeptide(s) of theinvention”) provides an unexpected decrease of 2 days in thetime-to-response. The time-to-response was objectified by the timenecessary for the recovery of platelets to ≥150,000/μl. In addition, theinvention provides an unexpectedly sustained and prolonged effect,reduced exacerbations, reduced hospitalization, reduced morbidity, areduced number of required PEs, reduced ischaemia, reduced organ damageand reduced death toll.

Therefore, the invention relates to the use of the polypeptides of theinvention to treat or ameliorate a vWF-related disease in a patient byan unexpectedly large decrease in the time-to-response, demonstrated byan accelerated platelet recovery. The invention also provides for lessfrequent PEs, while still maintaining the platelet recovery in the humanpatient at unexpectedly prolonged periods of time. Accordingly, methodsare provided for decreasing the time-to-response in a human patient byadministering to the patient a polypeptide of the invention, wherein theamount of the polypeptide administered is effective to change one ormore disease markers of TTP, such as the number of platelets,thrombocytopenia, neurocognitive function, disintegrin-like andmetalloprotease with thrombospondin repeats 13 (ADAMTS13) levels andanti-ADAMTS13 antibody titres, ADAMTS13 activity levels, cardiac marker(Troponin T or Troponin I), BNP (brain natriuretic peptide) orN-terminal pro brain natriuretic peptide (NT proBNP), and Brain damagemarkers (such as NSE (neuron specific enolase) and Sβ100 (S100beta)),preferentially an increase in the number of platelets.

In addition, the polypeptide of the invention when administered to ahuman TTP patient was safe as examined by safety laboratory markers,such as RICO, vWF and FVIII chromogene. Although there was a potentialfor an increased bleeding risk, this was wholly manageable.

The markers can be measured using standard methods known to and used bythe skilled person, such as various immunologically based assays,including enzyme-linked immunosorbent assays (ELISA; also known as anenzyme immunoassay (EIA)), radioimmunoassays or immunoenzymetric assays.Chemical, colorimetric and enzymatic based assays also may be used whensuitable.

Accordingly the present invention provides a polypeptide comprising atleast one ISVD against vWF for use in treating a vWF-related disease ina human in need thereof, by administering to the human a 5-40 mg dose ofsaid polypeptide, wherein said dose is followed within 15 min to 4 h bya first Plasma Exchange (PE).

The polypeptides of the invention were administered as adjunctivetreatment at specific times relative to the PE procedures to treat orprevent (e.g., reduce or ameliorate one or more symptoms associatedwith) a vWF-related disease, e.g., TTP.

The term “treating” refers to administering a therapy in an amount,manner, and/or mode effective to improve a condition, symptom, orparameter associated with a disease or to prevent progression of adisease, to either a statistically significant degree or to a degreedetectable to one skilled in the art. In the case of therapeutic use,the treatment may improve, cure, maintain, or decrease duration of, thedisease or condition in the subject. In therapeutic uses, the subjectmay have a partial or full manifestation of the symptoms. In a typicalcase, treatment improves the disease or condition of the subject to anextent detectable by a physician, or prevents worsening of the diseaseor condition. For instance, the clinical features and signs in an acuteepisode of TTP as depicted in Table 1 or as provided in the TTPtreatment guidelines (Scully et al. 2012 supra) improve. For instance,due to the treatment, the platelet count normalizes, the ADAMTS13autoantibody titre decreases and/or the ADAMTS13 activity increases, allas known in the art and/or further detailed herein (cf. infra). Aneffective amount, manner, or mode can vary depending on the subject andmay be tailored to the subject.

As used herein, the term “preventing” means to mitigate a symptom of thereferenced disorder. In particular, said term encompasses the completerange of therapeutically positive effects of administrating apolypeptide of the invention to a subject including reduction of,alleviation of, and relief from, a vWF related disorder, e.g. TTP, andsymptoms thereof. The term “prevention” includes the prevention orpostponement of development of the disease, prevention or postponementof development of symptoms and/or a reduction in the severity of suchsymptoms that will or are expected to develop. These further includeameliorating existing symptoms, preventing additional symptoms andameliorating or preventing the underlying causes of symptoms.

As used herein, the terms “subject” and “patient” are usedinterchangeably. As used herein, the terms “subject” and “subjects”refer to an animal, e.g., a mammal including a non-primate (e.g., a cow,pig, horse, donkey, goat, camel, cat, dog, guinea pig, rat, mouse,sheep) and a primate (e.g., a monkey, such as a cynomolgus monkey,gorilla chimpanzee and a human). A “patient” preferably refers to ahuman. Said patient can include elderly, adults, adolescents andchildren, from any age, for instance children ranging from the age of 2years to less than 12 years, adolescents ranging from 12 years to lessthan 18 years, adults ranging from 18 years to less than 65 years, andelderly from 65 years and up,

Non-limiting examples of vWF-related diseases that can be treatedinclude, but are not limited to, e.g. acute coronary syndrome (ACS),transient cerebral ischemic attack, unstable or stable angina pectoris,stroke, myocardial infarction, thrombotic thrombocytopenic purpura (TTP)and Upshaw-Schulman syndrome, preferably TTP.

The PE procedures to treat or prevent a vWF-related disease, such ase.g., TTP have been described in the Guidelines on the diagnosis andmanagement of TTP and other thrombotic microangiopathies (Scully et al.2012 supra), which is explicitly incorporated herein by reference.Complete remission is defined as normal platelet count, i.e.≥150,000/μl, and optionally the absence of exacerbations (cf. Scully etal. 2012, supra).

As used herein the “time-to-response” is the time between the firsttreatment of a patient having an acute TTP episode and a platelet countof ≥150,000/μl, in which the first treatment is a PE or theadministration of a polypeptide of the invention, or both, whichever isthe earliest.

The term “Plasma exchange” (“PE”) refers to a therapeutic procedure usedto treat a variety of diseases, including TTP, through the bulk removalof plasma, i.e. a procedure in which a large volume of plasma isremoved, usually 1-1.5 plasma volumes, which is replaced with areplacement fluid (Winters 2012 Hematology ASH Education Book 1:7-12).Through the bulk removal and replacement of plasma, PE removespathologic substances such as auto antibodies against ADAMTS13 andULvWF, but also some platelets. Plasma is used as a replacement fluid toreplace ADAMTS13 when treating thrombotic thrombocytopenic purpura(McLeod Best Pract Res Clin Haematol. 2006; 19:157-167). The bulkremoval and replacement of plasma also has implications for laboratorytesting, making patient testing intricate.

Because PE involves the bulk removal of plasma, anything circulating inthe plasma will be removed. Hence, this procedure is nonselective,removing both normal and pathologic plasma components, but also anymedicaments to treat TTP administered before PE.

The person skilled in the art is well acquainted in determining thenumber of platelets. Platelet counts can be done by any method known inthe art, such as manually using a hemocytometer or with an automatedanalyzer, e.g. electronic counting. Counts can also be estimated duringblood smear examination. The microscopic method uses a phase contrastmicroscope to view blood on a hemacytometer slide. Electronic countingof platelets is the most common method. There are two types ofelectronic counting, voltage-pulse and electro-optical counting systems.For instance, the ADVIA hematology analyzer can be used for obtainingplatelet counts and verify the obtained count by estimating counts on aWright's-stained blood smear. The ADVIA measures platelets by flowcytometry based on principles of light scattering. For instance,platelets are identified by their size (<30 FL, low angle light scatter)and refractive index (n=1.35 to n=1.40 or high angle light scatter).

In various patients following an acute episode of TTP, the polypeptideof invention comprising at least one ISVD against vWF, e.g. ALX 0081,was administered after said patient had received a PE (“preceding PE”; aPE preceding the administration of the first dose of the polypeptide ofthe invention). It was observed that in the group of subjects whichreceived a preceding PE (also indicated as “one PEX prior torandomization”), the median of the time-to-response was unexpectedlydecreased by 2 days from 4.31 days for the Placebo arm to 2.44 days forthe treatment arm: 43% reduction (Table 5; PEX prior toRandomization=YES).

Accordingly, the present invention relates to performing a PE (precedingPE) to a patient in need thereof, e.g. a patient with an acute episodeof TTP, followed by a next PE within 24 h of said preceding PE, andadministering a polypeptide of the invention (“first dose”) about 8 h, 7h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30 min, 20 min, 15 min, 10 minor even 5 min before starting said next PE, such as from 6 h to 15 minbefore starting said next PE (the “first PE”). In the present invention,the term “first dose” means the first administration of a polypeptide ofthe invention to a patient in need thereof, e.g. after an acute episodeor every acute episode of TTP.

In an embodiment, an administration of the polypeptide of the inventionto a patient, preferably a first dose is followed within 5 min to 8 h,such within 10 min to 6 h or 15 min to 4 h, for instance within 8 h, 7h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30 min, 20 min, 15 min, 10 minor even 5 min by a PE.

In the present invention, the term “first PE” means the first PEperformed after (or in some cases concurrent with) administration to apatient of a first dose of the polypeptide of the invention. Thepolypeptide of the invention can be administered or used foradministration in the form of a liquid solution (e.g., injectable andinfusible solutions). Such compositions can be administered by aparenteral mode (e.g., subcutaneous, intraperitoneal, or intramuscularinjection), or by inhalation. The phrases “parenteral administration”and “administered parenterally” as used herein mean modes ofadministration other than enteral and topical administration, usually byinjection, and include, subcutaneous (s.c.) or intramuscularadministration, as well as intravenous (i.v.), intracapsular,intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal,subcuticular, subcapsular, subarachnoid, intraspinal, epidural andintrasternal injection and infusion. Preferably the second or furtherdoses of the polypeptides of the invention described herein areadministered subcutaneously.

Preferably, the administration of the first dose of a polypeptide of theinvention following an acute episode of TTP is an intravenous bolusinjection, e.g. delivering the polypeptide through an intravenous line,administered all at once, over a period of a minute or two. Even morepreferably, the administration of the first dose of a polypeptide of theinvention following an acute episode of TTP is an intravenous pushinjection, e.g. delivering the polypeptide through an intravenous line,administered all at once, over a period of about 30 seconds or less.

It was surprisingly found that the polypeptides of the inventionadministered before PE (even without a preceding PE), in which timethere is no direct pharmacological targeting of the active process ofULvWF-mediated platelet aggregation and it can be expected that said PEremoves the polypeptide, were still able to reduce the median of thetime-to-response by an unexpectedly large decrease of 2 days from 4.92days for the Placebo arm to 3.00 days for the Caplacizumab arm: 39%reduction (Table 5: PEX prior to Randomization=NO).

The inventors considering that the polypeptide of the invention is safeto use as was demonstrated in previous studies in healthy volunteers andthe present study with TTP patients (cf. Example 7.5.3), that TTP mightbe hard to diagnose, especially acute bouts of TTP, and that any timelost before starting a treatment results in adversities, concluded thatthis finding has the benefit that a treatment with the polypeptide ofthe invention can already be started timely, even before the patiententers a hospital, such as e.g. in an ambulance. Preferably, thepolypeptide of the invention such as ALX 081 is administered by anintravenous push injection, since this can easily be performed outsidehospitals, thus saving valuable time.

Accordingly, the present invention relates to administering to a patientin need thereof, such as e.g. patients with acute episodes (acute bouts)of TTP, a polypeptide of the invention about 8 h, 7 h, 6 h, 5 h, 4 h, 3h, 3 h, 1 h, 45 min, 30 min, 20 min, 15 min, 10 min or even 5 min beforestarting PE, such as from 6 h to 15 min before starting PE (“firstdose”).

In an embodiment, the administration of a first dose of a polypeptide ofthe invention following an acute episode of TTP is followed by a PE(“first PE”). This first PE, whether or not preceded by a preceding PE,is followed by administration of a second or further dose of thepolypeptide of the invention (“second dose” or “further dose”).Preferably, the second dose or further dose is administered within 120,90 or 60 min, such as within 1-60 min, for instance, within 50, 45, 40,35, 30, 25, 20, 15, 10, 5, 4, 3, 2 or even 1 min after the first PE. Insome cases it may be advantageous to administer the second or furtherdose together or concurrently with the replacement fluid, e.g. theplasma of the PE.

In additional embodiments, a first dose, a second dose or further doseof the polypeptide of the invention is about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 40, 50, 60,70 or 80 mg, preferably 5-40 mg even more preferably 10 mg, which can beadministered to a patient in need thereof, preferably per day. Foradministration to juvenile patients, such as e.g. children andadolescents, the dose may be adjusted to the weight of the patient. Inparticular embodiments the dose is about 0.01, 0.025, 0.05, 0.075, 0.1,0.12, 0.14, 0.15, 0.16, 1.08, 0.2, 0.22, 0.24 or 0.25 mg/kg, preferably0.143 mg/kg which corresponds to a 10 mg dose in a 70 kg adult.

In an embodiment, the present invention relates to the administration ofabout 5 to 40 mg, preferably 10 mg of a polypeptide of the invention,e.g. ALX 0081, within 1-60 min after a PE procedure, e.g. the first PE,the second PE or a further PE.

In an embodiment, the polypeptide of the invention, e.g. ALX 0081, isadministered once per day or twice per day to a TTP patient in needthereof, preferably a patient with a platelet count below 100,000/μlplasma and/or a patient with an ADAMTS13 activity of ≤10% such as ≤5%:

In a further embodiment, a TTP patient in need thereof is treated with

-   (i) PE; and-   (ii) a dose of 5-40 mg preferably 10 mg of said polypeptide 60 min    to 1 min after said PE of step (I),    wherein step (i) and step (ii) are repeated once or twice per day    until the platelet count of said patient is at least 50,000/μl    plasma, such as 75,000, 100,000, 125,000 or even 150,000 per plasma.

In some cases it may be advantageous to repeat step (i) and step (ii)for a minimum of two days after complete remission (a platelet count of≥150,000/μl plasma).

In an embodiment, 5-40 mg of the polypeptide of the invention isadministered daily or twice daily for at least 5, 10, 15, 20, 25, 30,60, 90 or even 120 days after the platelet count of said patient is≥150.000/μl plasma, particularly when the ADAMTS13 activity of saidpatient is ≤10% such as ≤5%, or after the last PE.

When evaluating the data according to stratification (1 PEX prior toRandomization: YES & NO), an overall Hazard Ratio for the overallpopulation aggregates to 2.197 with a p value=0.013. This Hazard Ratiomeans that at any time, subjects receiving the polypeptide of theinvention have more than twice the rate of achieving the primaryendpoint of confirmed platelet recovery in comparison to subjects onPlacebo. In addition, this platelet recovery is achieved 2 days fasterin the treatment group.

Hence, the administration of polypeptides comprising at least one ISVDagainst vWF, such as ALX 0081, to human TTP patients following an acuteepisode of TTP provides an unexpected decrease in the time-to-response,independent of the order of administration of said polypeptide and saidPE, e.g. whether the PE is performed before or after the administrationof the first dose of the polypeptide of the invention.

It was further surprisingly found that the number of exacerbationsdecreased from 11 in the Placebo group to 3 in the Caplacizumab group.Hence, there are 3 times more exacerbations in the placebo group (i.e.TTP patients receiving PE and a placebo instead of the polypeptide ofthe invention) compared to the treatment group (i.e. TTP patientsreceiving PE and the polypeptide of the invention; also indicated asCaplacizumab group). The term “exacerbation” as used herein refers to arecurrent thrombocytopenia following a confirmed platelet response andrequiring a re-initiation of daily PE treatment after 1 day but 30 daysafter the last PE.

This indicates that the polypeptide of the invention, such as ALX 0081,can be solely responsible for treating and/or alleviating (the symptomsof) TTP.

Accordingly, the present invention relates to a polypeptide comprisingat least one ISVD against vWF, such as ALX 0081, for use in treating avWF-related disease, such as TTP, in a human in need thereof, byadministering to the human a dose of 1-80 mg or 5-40 mg, such as 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 50, 60, 70 or 80 mg, preferably 10 mg of said polypeptide.

Based on this surprising observation, a further optimized treatmentprotocol was designed by the present inventors, in essence based on theidea that the distribution of confirmed platelet response time isshorter and not skewed and biased to the right (longer time to response)in the CAP arm in comparison to the placebo arm. In the furtheroptimized treatment protocol, all subjects are treated with a fixed PEtreatment period, which is set for 3-5 days, such as 3 days or 4 days or5 days, preferably 3 days. In this case, the PE treatment period can beindependent of the recovery of platelets (≥150,000/μl). In the furtheroptimized treatment protocol, the burden for the patient and the costsare decreased.

Accordingly, the present invention relates to a polypeptide comprisingat least one ISVD against vWF, such as ALX 0081, for use in treating avWF-related disease in a human in need thereof, comprising: (i)performing a PE; and (ii) administering a dose of 5-40 mg, such as 10 mgof the polypeptide of the invention 15 min to 4 h after said PE of step(i), wherein step (i) and step (ii) are repeated once per day for 3-5days, such as 3 days, 4 days or 5 days, preferably 3 days; followed byfurther comprising administering once per day a dose of 5-40 mg, such as10 mg of said polypeptide for at least 10 days, such as at least 20 daysor at least 30 days and/or for at least 10 days, such as at least 20days or at least 30 days after the platelet count of said patient wasfor the first time ≥150.000/μl,

In the present study, for up to one year TTP patients have beenfollowed-up for remission. The term “remission” as used herein refers toas confirmed platelet response and the absence of exacerbation. The term“confirmed platelet response” as used herein refers to thetime-to-response of treatment as defined by a recovery of platelets≥150,000/μL, which response must be confirmed at 48 hours after theinitial reporting of platelet recovery above 150,000/μL by a de novomeasure of platelets ≥150,000/μl, and preferably LDH ≤2×ULN.

As demonstrated in herein (Example 7.5.5; Table 8), overall, 29 patientsin the treatment group went into remission, compared to 18 patients inthe placebo group. Hence, the treatment arm presents 1.6× more subjectswith complete remission versus the Placebo arm.

As noted above, the platelet count is the primary means for assessingremission. Measurement of ADAMTS13 activity in patients with a historyof classical TTP is important because low levels have been shown to bepredictive of relapse. However, it is unclear at present (and the datais conflicting) as to whether the titre of an inhibitory antibody toADAMTS13 is significant i.e. are those individuals with a high titreanti-ADAMTS13 antibody more likely to relapse than those with a lowtitre. The person skilled in the art appreciates that current tests ofADAMTS13 are performed under static conditions and do not alwaysaccurately reflect the physiological changes that occur in vivo(http://practical-haemostasis.com/Miscellaneous/Miscellaneous%20Tests/adamts13_assays.html).

The present inventors now unexpectedly observed that remission appearsmore pronounced for the subgroup of subjects with low Baseline ADAMTS13activity (i.e. less than 10%, such as less than 5%), when startingtreatment, e.g. administering the first dose, of the polypeptide of theinvention, such as ALX 0081 (cf. Example 7.5.8).

Accordingly, the present invention relates to a polypeptide comprisingat least one ISVD against vWF for use in treating a vWF-related diseasein a human in need thereof, by administering to said human a first doseof 1-40 mg, preferably 10 mg of said polypeptide, until the plateletcount of said human is ≥150000/μl. In a preferred aspect, said human hasan ADAMTS13 activity of less than 10%, such as less than 5% whenadministering said polypeptide.

In the present study, for up to one year TTP patients have beenfollowed-up for relapses. The term “relapse” as used herein refers to ade novo event of TTP that occurs later than 30 days after the last dailyPE, e.g. 0-2 days after the TTP patient showed complete remission. The“later than 30 days” date coincides with the last administration of thepolypeptide of the invention in the present study.

Although the polypeptide of the invention was not administered anymore,it has been found that the number of relapses in the Caplacizumab groupequaled the number of relapses in the Placebo group (see Table 7, inExample 7.5.4).

The inventors observed that in both treatment arms, relapses are moreprominent in patients with a baseline ADAMTS13 activity of <10%, such as<5%, even though the ADAMTS13 activity was only available in a subset ofthe patients. The inventors hypothesized (without being bound to anytheory) that this may indicate that patients with baseline ADAMTS13activity of <10%, such as <5% are more prone to relapses (orexacerbations), when stopping administration of the polypeptide of theinvention, such as ALX 0081.

In particular, the data support the use of ADAMTS13 activity aspredictive marker for recurrences of TTP and its potential for treatmentdecisions. ADAMTS13 activity is able to predict relapses which occurshortly after stopping caplacizumab treatment. These relapses areconsidered as relapses of the presenting TTP episode (unresolved diseaseactivity, based on continuously low ADAMTS13 activity). A 30-daytreatment period (post PE) with caplacizumab has demonstrated asignificant impact on the number of exacerbations. Hence, extending thecaplacizumab treatment period for those patients at risk for relapse(i.e. with underlying disease activity based on ADAMTS13 activity) willmaintain the protective effects of caplacizumab until the underlyingdisease is adequately treated and resolved. Conversely, precautionarytreatment with caplacizumab will reduce the risk of a—new—acute episodeof TTP.

Hence, treatment with polypeptide of the invention, such as ALX 0081,should be continued for longer periods compared to patients with higheractivity. The polypeptide of the invention should be administered to aTTP patient to reduce the risk of and/or prevent the chance ofrelapse(s) until the ADAMTS13 activity was at least 10%, such at least15%, 20%, 25%, 30%, 35%, 40%, 45%, or even 50% compared to the normal orreference activity.

Accordingly, the present invention relates to a polypeptide comprisingat least one immunoglobulin single variable domain (ISVD) against vonWillebrand Factor (vWF) for use in reducing the risk of and/orpreventing an acute episode of a vWF-related disease, e.g. TTP, in ahuman in need thereof, comprising a step (i): administering to saidhuman a dose of 5-40 mg, preferably 10 mg, of said polypeptide.Preferably, said risk is reduced by a factor of at least 1.2, 1.3, 1.4,1.5, 1.6, 1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10,or even more such as 20, 50 or even 100. Preferably said risk is reducedby 10% or even more such as 20%, 30%, 40%, 50%, 60% or more, such as 80%or even 100%.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said step (i) of administering to said human saidpolypeptide is repeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10times, or even more than 10 times, such as 20 times, preferably morethan 30 times or even more.

The method according to claim 1, wherein said step (i) of administeringto said human said polypeptide is repeated for at least 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 days, or even more than 10 days, such as 20 days,preferably more than 30 days, such as 2 months, 3 months, 4 months, 5months, 6 months or even more.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said dose is administered 1 time per day or two timesper day.

Accordingly, the present invention relates to a polypeptide as describedherein, further comprising

-   (ii) measuring the ADAMTS13 activity of said patient;-   (iii) comparing said ADAMTS13 activity with a reference ADAMTS13    activity; and-   (iv) if said ADAMTS13 activity is lower than 30%, such as 20%, 15%,    10% or 5% of said reference ADAMTS13 activity, then repeating said    step (i) of administering to said human said polypeptide.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said ADAMTS13 activity of said patient is measured everyday, or every 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, preferably at leastonce every week.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein step (i) is repeated until said ADAMTS13 activity is atleast 5%, 10%, 15%, such 20%, or even 30% or higher of said referenceADAMTS13 activity.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein step (i) of administering to said human said polypeptideis repeated until said ADAMTS13 activity is at least 5%, 10%, 15%, suchas 20% or 30% of said reference ADAMTS13 activity on at least 2consecutive measurements. Preferably, said 2 consecutive measurementsare at least 24 h, more preferably 48 h apart, such as at least 3 daysapart, or even more such as, 4, 5, 6, or even 7 days apart, preferably aweek apart.

Accordingly, the present invention relates to a polypeptide as describedherein, wherein said step (i) of administering to said human saidpolypeptide is repeated for at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9,or 10 days, or even more than 10 days, such as 20 days, preferably morethan 30 days or even more, after said ADAMTS13 activity is at least 5%,at least 10%, at least 15%, such as 20% or at least 30% of saidreference activity on at least 2 consecutive measurements.

Accordingly, the present invention relates to a polypeptide comprisingat least one immunoglobulin single variable domain (ISVD) against vonWillebrand Factor (vWF) for use in reducing the risk of and/orpreventing an acute episode of a vWF-related disease, such as TTP, in ahuman in need thereof, comprising step (i): administering to said humana dose of 5-40 mg, preferably 10 mg of said polypeptide, furthercomprising

-   -   measuring the ADAMTS13 activity of said patient;    -   comparing said ADAMTS13 activity with a reference ADAMTS13        activity; and    -   if said ADAMTS13 activity is ≥5%, such as ≥10%, or even ≥15%, or        more than 20% or 30% of said reference ADAMTS13 activity, then        repeating said step (i) for at most 30 days, such as at most 20        days, or even 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 days or even 1 day.

Accordingly, the present invention relates to a polypeptide comprisingat least one immunoglobulin single variable domain (ISVD) against vonWillebrand Factor (vWF) for use in reducing the risk of and/orpreventing an acute episode of a vWF-related disease, such as TTP, in ahuman in need thereof, comprising at least the following steps:

-   (i) measuring the ADAMTS13 activity of said patient;-   (ii) comparing said ADAMTS13 activity with a reference ADAMTS13    activity; and-   (iii) if said ADAMTS13 activity is lower than 30%, 20%, 15%, 10% or    5% of said reference activity, then administering to said human a    dose of 5-40 mg, preferably 10 mg of said polypeptide comprising at    least one immunoglobulin single variable domain (ISVD) against von    Willebrand Factor (vWF).

As used herein, reducing risk or incidence includes decreasing theprobability or incidence of an indication, symptom or result ofvWF-related disease, such as TTP, for a subject compared to a relevant,e.g. untreated, control population, or in the same subject prior totreatment according to the invention.

An indication, symptom or result of a vWF-related disease, such as TTP,as used herein includes organ damage, ischaemic damage, microthrombiformation, exacerbations, mortality, relapses, one or more diseasemarkers of a vWF related disease, such as TTP, include the number ofplatelets, thrombocytopenia, neurocognitive function, ADAMTS13 levelsand anti-ADAMTS13 antibody titres, ADAMTS13 activity levels, cardiacmarker (Troponin T or Troponin I), BNP (brain natriuretic peptide) orN-terminal pro brain natriuretic peptide (NT proBNP), creatinine, andBrain damage markers (such as NSE (neuron specific enolase) and Sβ100(S100beta)), preferentially organ damage markers, such as LDH levels,troponin T and/or troponin I levels, and/or creatinine levels.

The reduced risk or incidence can include delaying or preventing theonset of an indication, symptom or result of vWF-related disease, suchas TTP. Risk or incidence can also be reduced if the severity of anindication, symptom or result of vWF-related disease, such as TTP, isreduced to a level such that it is not of clinical relevance. That is,the indication, symptom or result of a vWF-related disease, such as TTP,may be present but at a level that does not endanger the life,activities, and/or wellbeing of the subject. In some circumstances theoccurrence of the vWF-related disease, such as TTP, is reduced to theextent that the subject does not present any signs of the vWF-relateddisease, such as TTP, during and/or after the treatment period.

It will be appreciated that no actual proof of reduced risk for anindividual can be obtained because if treatment is provided then itcannot be said whether an indication, symptom or result of a vWF-relateddisease, such as TTP, would have occurred, or would have occurred soonerin the absence of such treatment. Thus, the concept of risk and,increased or reduced risk refer to statistical values only. Further,reduction of risk of an indication, symptom or result of a vWF-relateddisease, such as TTP, can be reflected in a reduction in the severity ofan indication, symptom or result of a vWF-related disease, such as TTP,as well as in the absence of observation or delay in observation of anindication, symptom or result of a vWF-related disease, such as TTP.

It will be appreciated that the polypeptide of the invention reduces therisk of and/or preventing an acute episode of a vWF-related disease,such as TTP. Hence, the indication, symptom or result of a vWF-relateddisease, such as TTP, is also reduced. Given the pathophysiology ofacquired TTP whereby ULvWF strings consume platelets in the formation ofmicrothrombi, it was reasoned that the recovery of platelet counts is anindirect measure of prevention of further microthrombi formation. Themorbidity and the acute mortality associated with acquired TTP is aresult of these microthrombi.

Indeed, this reasoning is supported by the normalization of organ damagemarkers. In particular, the results indicate that the organ damagemarkers, such as troponin I and T, LDH and creatinine, return faster tonormal levels in subjects receiving the polypeptide of the invention,e.g. ALX 0081, than in subjects receiving placebo (cf. Example 7.5.7).

Hence, the results suggest that a faster normalization rate of theseorgan damage markers is linked to a better clinical outcome, i.e. areduced risk of and less organ damage due to organ ischemia caused bymicrothrombi.

Accordingly, the present invention relates to a method as describedherein, wherein

-   -   the risk of organ damage, ischaemic damage and/or microthrombi        formation is reduced by 10%, 20%, 30%, preferably by at least        40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even to        100% (e.g. absence of organ damage, ischaemic damage and/or        microthrombi formation due to the vWF-related disease);    -   the risk of organ damage, ischaemic damage and/or microthrombi        formation is reduced by a factor 1.2, 1.3, 1.4, 1.5, 1.6, 1.75,        1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even        more such as 20, 50 or even 100;    -   organ damage, ischaemic damage and/or microthrombi formation is        reduced preferably by at least 10%, 20%, 30%, 40%, or even at        least 50%, such as 60%, 70%, 80%, 90% or even to 100%;    -   organ damage, ischaemic damage and/or microthrombi formation is        reduced by a factor 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or        more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more such        as 20, 50 or even 100;    -   organ damage markers, such as LDH levels, troponin T, troponin I        levels, and/or creatinine levels, return to at least 40%, or        even at least 50%, such as 60%, 70%, 80%, 90% or even to 100% of        normal levels;    -   organ damage markers, such as LDH levels, troponin T, troponin I        levels, and/or creatinine levels, improve by at least 20%, such        30% or even higher, such as 40%, or even at least 50%, such as        60%, 70%, 80%, 90% or even to 100% of normal levels. Preferably,        said organ damage, such as LDH levels, troponin T, troponin I        levels, and/or creatinine levels, markers improve in less than        30 days of treatment, preferably, in less than 20 days of        treatment, such as, less than 15, 10, 9, 8, 7, 6, 5, 4, 3, 2        days or even within 1 day.    -   the number of platelets is kept at ≥150000/μl.    -   the time to platelet normalization (≥150000/μl) is reduced by at        least 10%, 20%, 30%, 35%, 39%, preferably by at least 40%, or        even at least 50%, such as 60%, 70%, 80%.    -   the risk of exacerbations is reduced by at least 10%, 20%, 30%,        40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even to        100%;    -   the risk of exacerbations is reduced by a factor, 2 or more,        such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more such as        20, 50 or even 100;    -   mortality due to said vWF related disease is reduced by 10%,        20%, 30%, preferably by at least 40%, or even at least 50%, such        as 60%, 70%, 80%, 90% or even to 100%;    -   mortality due to said vWF related disease is reduced by a        factor, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or        even more such as 20, 50 or even 100; and/or    -   remission is increased by a factor 1.2, 1.3, 1.4, 1.5, 1.6,        1.75, 1.8, 2 or more, such as 3, 4, 5, 6, 7, 8, 9, or even 10,        or even more such as 20, 50 or even 100.

The term “reference activity” as used herein, refers to the averageADAMTS13 activity of 5 healthy subjects in the assay performed, which isset at 100%. For instance, in a FRETS-vWF73 assay, the calibration curvegenerated using a normal human plasma pool, in which the slope of theregression curve is calculated for each calibration sample, and used togenerate the calibration curve (trend line: y=ax+b; with x=ADAMTS13(%)and y=delta RFU/delta time). The ADAMTS13 activity (%) of a sample isthen calculated as: (y− b)×1/a. Indeed, in general the patients thatrelapsed had a lower ADAMTS13 activity than the patients who did notrelapse,

Accordingly, the present invention relates to a polypeptide for reducingthe risk of and/or preventing ischaemic damage, organ damage and/ormicrothrombi formation, for instance causable by a vWF-related disease,such as TTP, in a human in need thereof, comprising at least thefollowing step (i) administering to said human a dose of 5-40 mg/day,preferably 10 mg/day of a polypeptide comprising at least oneimmunoglobulin single variable domain (ISVD) against von WillebrandFactor (vWF); wherein administration of said polypeptide reduces therisk of and/or prevents ischaemic damage, organ damage and/ormicrothrombi formation by at least 10%, 20%, 30%, preferably by at least40%, or even at least 50%, such as 60%, 70%, 80%, 90% or even to 100%.Preferably, administration of said polypeptide reduces the risk ofand/or prevents ischaemic damage, organ damage and/or microthrombiformation by a factor 1.2, 1.3, 1.4, 1.5, 1.6, 1.75, 1.8, 2 or more,such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more such as 20, 50 oreven 100.

Accordingly, the present invention relates to a polypeptide for reducingthe risk of and/or preventing ischaemic damage, organ damage and/ormicrothrombi formation as described herein, wherein said step (i) ofadministrating said polypeptide is repeated for at least 1, 2, 3, 4, 5,6, 7 days, or even longer such as 1 week, 2 weeks, 3 weeks, or evenlonger such as 1 month or even 2 months.

Accordingly, the present invention relates to a polypeptide for reducingthe risk of and/or preventing ischaemic damage, organ damage and/ormicrothrombi formation as described herein, further comprising measuringADAMTS13 activity of said patient, preferably once per week.

Accordingly, the present invention relates to a polypeptide for reducingthe risk of and/or preventing ischaemic damage, organ damage and/ormicrothrombi formation as described herein, wherein said step (i) ofadministrating said polypeptide is repeated for at least 1, 2, 3, 4, 5,6, 7 days, or even longer such as 1 week, 2 weeks, 3 weeks, or evenlonger such as 1 month or even 2 months when the ADAMTS13 activity is[for the first time]≥5%, such as ≥10%, or even ≥15% of a referenceADAMTS13 activity.

Accordingly, the present invention relates to a polypeptide of theinvention for treating a symptom of a vWF-related disease, such as TTP,in a human suffering from said disease, comprising administering to thesubject a polypeptide of the invention, in an amount effective to treatthe symptom of a vWF-related disease in a human suffering from saiddisease.

Accordingly, the present invention relates to a polypeptide of theinvention for inhibiting in a human the onset or progression of avWF-related disease, such as TTP, the inhibition of which is effected bybinding of a polypeptide comprising at least one immunoglobulin singlevariable domain (ISVD) against von Willebrand Factor (vWF) to vWF,comprising administering to the human at a predefined interval effectiveinhibitory doses of said polypeptide, wherein each administration of theantibody delivers to the human from 0.1 mg per kg to 25 mg per kg of thehuman's body weight, so as to thereby inhibit the onset or progressionof the disease in the human.

Accordingly, the present invention relates to a polypeptide for reducingthe likelihood of a human contracting ischaemic organ damage by avWF-related disease, which comprises administering to the human at apredefined dose a polypeptide comprising at least one immunoglobulinsingle variable domain (ISVD) against von Willebrand Factor (vWF),wherein each administration of the antibody delivers to the human from0.1 mg per kg to 25 mg per kg of the human's body weight, so as tothereby reduce the likelihood of the human contracting ischaemic organdamage.

Modeling based on these results indicates that maintaining administeringpolypeptides for prolonged times of the invention would be efficaciousin preventing acute episodes. This advantageous profile results in adecreased health hazard. Hence, it can be concluded that the polypeptideof the invention prevents relapses.

Accordingly, the present invention relates to administering thepolypeptide of the invention every 1, 2, 3, 4, 5, 6 7 days or even 2, 4,6, or 8 weeks at doses ranging from 1-80 mg, such as 5-40 mg, preferablyin preventing acute episodes of TTP. Particular efficacious doses are10-20 mg. In particular embodiments, the dose comprises about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 50, 60, 70 or 80 mg, preferably 10 mg of a polypeptidecomprising at least one ISVD against vWF, such as ALX 0081.

In an embodiment the present invention relates to a method of preventingrelapse in a TTP patient, comprising

-   (1) measuring ADAMTS13 activity from a TTP patient by an assay, such    as a direct or an indirect assay;-   (2) comparing said activity of step (1) with a reference value    (normal value); and-   (3) if the ADAMTS13 activity of step (1) is less than 15%, such as    less than 10% and less than 5%, of the reference value, then    administering the polypeptide of the invention, e.g. ALX 0081,    thereby preventing relapse.

Preliminary results suggest that administration of the first dose of thepolypeptide of the invention before the first PE already results in anincrease in the number of platelets.

Accordingly, the present invention relates to administering thepolypeptide of the invention in a patient in need thereof, such as e.g.a patient experiencing an acute episode of TTP, a dose of about 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 50, 60, 70 or 80 mg, preferably 10 mg of a polypeptidecomprising at least one ISVD against vWF, such as ALX 0081.

The polypeptides of invention comprising at least one ISVD against vWF,e.g. ALX 0081, can be administered to a subject (e.g., a human subject)alone or combination with a second agent, e.g., a second therapeuticallyor pharmacologically active agent, to treat or prevent (e.g., reduce orameliorate one or more symptoms associated with) a vWF-related disease,e.g., TTP.

Non-limiting examples of agents that can be co-formulated with thepolypeptides of invention comprising at least one ISVD against vWF, e.g.ALX 0081, include, for example, adjunctive immunosuppressive treatment(e.g. corticosteroids such as (methyl)prednisolone or(methyl)-prednisone; or rituximab), antiplatelet agents (e.g. aspirin),supportive therapy with red cell transfusion or folate supplementation,treatment with vincristine or cyclosporin, anti-autoADAMTS13 antibodies,or ADAMTS13. Such combination therapies may advantageously utilize lowerdosages of the administered therapeutic agents, thus avoiding possibletoxicities or complications associated with the various monotherapies.

In an embodiment, the present invention relates to a combination therapyof the polypeptide of the invention together with an immunosuppressivetreatment, in particular rituximab, which efficiently prevents relapsesin TTP patients. Preferably, the combination therapy is provided untilthe ADAMTS13 activity is at least ≥5%, such as ≥10%, >15%, >20%, 25%,30%, 35%, 40%, 45% or even normalised such as ≥50% of the normalactivity,

TTP remains a diagnosis based on clinical history, examination of thepatient and the blood film. ADAMTS13 assays help to confirm thediagnosis and monitor the course of the disease and possible need foradditional treatments. Acute episodes of TTP can be diagnosed accordingto Table 1 and the guidelines of, for instance, Scully et al. (2012supra)

TABLE 1 Clinical features and signs in acute episode of TTP.Thrombocytopenia Epistaxis, bruising, petechiae, gingival bleeding,haematuria, menorrhagia, gastrointestinal bleeding, retinal haemorrhageand haemoptysis Central neurological - Confusion, headache, paresis,aphasia, often flitting and dysarthria, visual problems, variable 70-80%encephalopathy, coma (10%) Fever (>37.5° C.) Non-specific symptomsPallor, jaundice, fatigue, arthralgia or myalgia Jaundice Resulting frommicroangiopathic haemolytic anaemia Renal Impairment Proteinuria,microhaematuria Cardiac Chest pain, heart failure, hypotensionGastro-intestinal tract Abdominal pain

The efficacy of any particular polypeptide of the invention or dosingregimen may be determined by methods available to those of skill in theart. Briefly, during a clinical trial, the patients may be observed bymedical personnel and the state of disease is assessed by anycombination of criteria. The improvement of a patient's disease state isdetermined based on these criteria at numerous time points and thecombination of these determinations on a patient population is plottedto assess the efficacy of treatment.

In exemplary embodiments, assessment of efficacy may be measured by anyor all of the criteria set forth below:

-   -   Time-to-response of treatment, defined by a recovery of        platelets ≥150,000/μL. This response must be confirmed at 48        hours after the initial reporting of platelet recovery above        150,000/μL by a de novo measure of platelets ≥150,000/μL and        preferably by LDH ≤2×ULN    -   Number of subjects with complete remission    -   Number of (subjects with) exacerbations of TTP and time to first        exacerbation of TTP. Exacerbation is defined as recurrent        thrombocytopenia following a response and requiring a        re-initiation of daily PE treatment after ≥1 day but ≤30 days        after the last daily PE.    -   Number of subjects relapsing of TTP (defined as de novo event of        TTP that occurs later than 30 days after the last daily PE) for        a maximum of 1 year, and time to first relapse of TTP    -   Daily PE data, including serious adverse events (SAEs) related        to daily PE treatment    -   Neurocognitive function, as measured by a neurocognitive test        battery, at complete remission and at 1 year follow up. This        test will be preceded by the Glasgow Coma Score to measure the        state of consciousness of the subject    -   Improvement of organ dysfunction and improvement of TTP related        signs and symptoms    -   Total mortality within the daily PE treatment period and within        the subsequent study drug treatment period (including tapering)    -   Determination of biomarkers of TTP including but not limited to        a disintegrin-like and metalloprotease with thrombospondin        repeats 13 (ADAMTS13) levels and anti-ADAMTS13 antibody titres.

The person skilled in the art is familiar to determine the efficacies.

For instance, ADAMTS13 activity can be evaluated using electrophoresisof vWF multimers to detect ultra-large multimers uncleaved by theprotease (Moake et al. (1982) The New England journal of medicine 307,1432-1435; Furlan, et al. (1997) Blood 89, 3097-3103 7, 8). ADAMTS13activity can be tested employing FRETS-vWF73, a fragment of vWFchemically modified to emit fluorescence when cleaved by ADAMTS13. Inthe assay, FRETS-vWF73 is added to a sample of the patient's plasma, andthe change in fluorescence is measured over time to determine ADAMTS13activity. If an inhibitor is present, it is frequently a neutralizingIgG antibody directed against ADAMTS13, which can be measured by ELISA(Kokame et al. (2005) British journal of haematology 129, 93-100).Alternatively or in addition, ADAMTS13 activity can be determined asdescribed in for instance Vesely et al. (2003, supra), Fontana et al.(2004, supra) or Remuzzi et al. (Blood 2002; 100: 778-7852002). Forinstance, indirect ADAMTS13 activity assays involve the detection ofcleavage of products either of a full-length VWF molecule or a VWFfragment that encompasses the ADAMTS13 cleavage site in the A2 domain ofVWF. (1) Collagen Binding Assays. Normal plasma or purified VWF isincubated with the test plasma sample in the presence of BaCl2 and 1.5Murea which denatures the VWF. VWF is cleaved by ADAMTS13 and residualVWF is measured by its binding to collagen Type III. The bound VWF isquantitated using an ELISA assay with a conjugated anti-VWF antibody.(2) Ristocetin-Induced Aggregation. This is similar to thecollagen-binding assay above but residual VWF is measured byristocetin-induced platelet aggregation using a platelet aggregometer.(3) Functional ELISA assays. In this assay, a recombinant VWF fragmentis immobilised onto an ELISA plate using an antibody to a tag on theVWF. The VWF fragment encodes the A2 domain and the ADAMTS13 cleavagesite at Tyr1605-Met1606 and is tagged with S-transferase [GST]-histidine[GST-VWF73-His]. Plasma is added to the immobilised GST-VWF73-Hisfragment and cleavage of the immobilised fragment occurs at the ADAMTS13cleavage site. The residual, cleaved VWF fragment is measured by using asecond monoclonal antibody that recognises only the cleaved VWF fragmentand not the interact fragment. ADAMTS13 activity is, therefore,inversely proportional to the residual substrate concentration. Thismethod forms the basis for the TECHNOZYM® ADAMTS13 Activity ELISA.

The person skilled in the art is familiar in determining autoantibodiesagainst ADAMTS13, for instance, anti-ADAMTS13 autoantibodies can bedetermined by ELISA, such as the TECHNOZYM® ADAMTS13 INH ELISA(Technoclone).

The person skilled in the art is familiar in determining of RistocetinCofactor activity in human samples, for instance, the RistocetinCofactor can be determined by the vW Select of Bio/Data corp. on anaggregometer PAP-8E analyzer (Bio/Data corp.).

The person skilled in the art is familiar in determining Factor VIII inhuman samples, for instance using Coamatic Factor VIII (Chromogenix) ona STA-R evolution analyzer (Diagnostica Stage).

The person skilled in the art is familiar in determining von WillebrandFactor antigen in human samples, for instance using aimmunoturbidometric assay (e.g. using a STA Lia test vWF:Ag) on a STA-Revolution analyzer (Diagnostica Stago).

The person skilled in the art is familiar in determining LDH levels.Most methods are based on a lactate dehydrogenase-based enzymaticanalysis on a spectrophotometer. A convenient review is provided byMedbø et al. (2000) “Examination of four different instruments formeasuring blood lactate concentration”. Scand J Clin Lab Invest60:367-380. Various companies provide assays, such as Abnova (CatalogNumber KA1653) which measures the catalysis by LDH of theinterconversion of lactate and pyruvate, i.e. a non-radioactivecolorimetric LDH assay based on the reduction of the tetrazolium saltMTT in a NADH-coupled enzymatic reaction to a reduced form of MTT whichexhibits an absorption maximum at 565 nm. The intensity of the purplecolor formed is directly proportional to the enzyme activity. Similarly,in the Sigma Aldrich kit (MAK066-1KT), LDH reduces NAD to NADH, which isspecifically detected by colorimetric (450 nm) assay. Normal values areprovided in the Table 1.1 below.

The person skilled in the art is familiar in determining troponin I andT. In general, troponin T and I are measured by immunoassay methods,which are available on many different immunoassay platforms, e.g. DPCImmulite, Abbott AxSYM, Bayer ACS:Centaur, Ortho Vitros, Roche Elecsys,third generation. A convenient review is provided by Wu et al. (1999)National Academy of Clinical Biochemistry Standards of LaboratoryPractice: recommendations for the use of cardiac markers in coronaryartery diseases. Clin Chem. July 1999; 45(7):1104-21. Normal values areprovided in the Table 1.1 below.

The person skilled in the art is familiar in determining creatinine. Aconvenient review is provided by Peake and Whiting “Measurement of SerumCreatinine—Current Status and Future Goals” Clin Biochem Rev. 2006November; 27(4): 173-184. For instance, creatinine levels can bedetermined by Abcam Creatinine Assay Kit (ab65340) or BioVision'sCreatinine Assay Kit. In the assay, creatinine is converted to creatineby creatininase, creatine is converted to sarcosine, which isspecifically oxidized to produce a product which reacts with a probe togenerate red color (λmax=570 nm) and fluorescence (Ex/Em=538/587 nm).Normal values are provided in the Table 1.1 below. Since the amount ofcreatinine in the blood increases with muscle mass, men usually havehigher creatinine levels than do women.

TABLE 1.1 normal values Test Specimen Conventional Units SI UnitsCreatinine Serum 0.7-1.3 mg/dL 61.9-115 μmol/L Lactate Serum 60-160 U/L1-1.67 μkat/L dehydrogenase (LDH) Troponin I Plasma <0.1 ng/mL <0.1 μg/LTroponin T Serum ≤0.03 ng/mL ≤0.03 μg/L

It will be appreciated that the normal values provided in Table 1.1 canvary from lab to lab, between men and women, and by age. Nevertheless,the person skilled in the art will consider that depending on the assayused, the normal values provided by the manufacturer can normally beused as a reference, or alternatively, the normal values as assessed bythe clinician in the specific setting.

The polypeptides of the invention typically comprise at least one ISVDagainst vWF. The immunoglobulin single variable domains of the presentinvention bind to and/or have affinity for vWF. In the context of thepresent invention, “vWF” includes, but is not limited, to cynomolgus,baboon, pig, guinea pig, mouse, and/or human vWF and most preferredhuman vWF, i.e. SEQ ID NO: 20 or Gen Bank entry: NP_000543.

Preferably, the ISVD against vWF essentially consists of 4 frameworkregions (FR1 to FR4, respectively) and 3 complementarity determiningregions (CDR1 to CDR3, respectively), in which:

a) CDR1 comprises or essentially consists of:

-   -   the amino acid sequence YNPMG; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence YNPMG;        and        b) CDR2 comprises or essentially consists of:    -   the amino acid sequence AISRTGGSTYYPDSVEG; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AISRTGGSTYYPDSVEG; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AISRTGGSTYYPDSVEG;        and        c) CDR3 comprises or essentially consists of:    -   the amino acid sequence AGVRAEDGRVRTLPSEYTF; or    -   an amino acid sequence that has at least 80%, preferably at        least 90%, more preferably at least 95%, even more preferably at        least 99% sequence identity with the amino acid sequence        AGVRAEDGRVRTLPSEYTF; or    -   an amino acid sequence that has 2 or only 1 amino acid        difference(s) with the amino acid sequence AGVRAEDGRVRTLPSEYTF.

Even more preferably, the ISVD against vWF essentially consists of 4framework regions (FR1 to FR4, respectively) and 3 complementaritydetermining regions (CDR1 to CDR3, respectively), in which:

a) CDR1 is YNPMG (SEQ ID NO: 20); b) CDR2 is AISRTGGSTYYPDSVEG (SEQ IDNO: 21); and c) CDR3 is AGVRAEDGRVRTLPSEYTF (SEQ ID NO: 22).

Even more preferably, the ISVD against vWF is represented by SEQ ID NO:19 (12A02H1).

Preferably, the polypeptides of the invention comprise or consist of atleast two ISVDs against vWF.

Even more preferably, the polypeptides of the present invention compriseor consist of two ISVDs against vWF defined by SEQ ID NO:s 1-18, andmost preferably SEQ ID NO: 1 (ALX 0081; INN Caplacizumab). ALX 0081 is abivalent Nanobody, consisting of two identical monovalent buildingblocks, that target vWF.

The polypeptides comprising at least one ISVD against vWF, e.g. SEQ IDNO:s 1-19, may be used in a treatment of a vWF-related disease, inparticular thrombotic thrombocytopenic purpura (TTP).

The terms “polypeptide” and “amino acid sequence” are usedinterchangeably herein.

Thus, for example, suitable polypeptides for use in the invention mayinclude the compounds in Table A-1, e.g. SEQ ID NO: 1-19 or 20-22, or acompound having 80% or more, more preferably 85% or more, most preferred90%, 95%, 96%, 97%, 98%, 99% or more, amino acid sequence identity to acompound in Table A-1 (see Definition section for “sequence identity”).

Preferably the ISVD against vWF for use in the polypeptides of theinvention are 12A02H1-like compounds. For the purposes of the presentdescription a 12A02H1-like compound is a compound which comprises12A02H1 (i.e. SEQ ID NO: 19) or a compound having 80% or more, morepreferably 85% or more, most preferably 90%, 95%, 96%, 97%, 98%, 99% ormore, amino acid sequence identity to 12A02H1 (SEQ ID NO: 19). Aparticularly preferred polypeptide comprising two ISVDs against vWF isALX 0081 (SEQ ID NO: 1).

Immunoglobulin single variable domains, such as camelid VHH domains,camelized VH domains or humanized VHH domains, represent a rapidlygrowing class of therapeutics. For example, immunoglobulin singlevariable domains against vWF have been described in WO2004/015425,WO2004/062551, WO2006/074947, WO2006/122825, WO2009/115614, andWO2011/067160. Further preferred immunoglobulin single variable domainsfor use in the polypeptides of the invention include the improvedNanobodies described in WO06/122825.

Unless indicated otherwise, the term “immunoglobulin sequence”—whetherused herein to refer to a heavy chain antibody or to a conventional4-chain antibody—is used as a general term to include both the full-sizeantibody, the individual chains thereof, as well as all parts, domainsor fragments thereof (including but not limited to antigen-bindingdomains or fragments such as VHH domains or VH/VL domains,respectively). In addition, the term “sequence” as used herein (forexample in terms like “immunoglobulin sequence”, “antibody sequence”,“variable domain sequence”, “VHH sequence” or “protein sequence”),should generally be understood to include both the relevant amino acidsequence as well as nucleic acids or nucleotide sequences encoding thesame, unless the context requires a more limited interpretation.

The term “immunoglobulin single variable domain” (“ISVD”),interchangeably used with “single variable domain”, defines moleculeswherein the antigen binding site is present on, and formed by, a singleimmunoglobulin domain. This sets immunoglobulin single variable domainsapart from “conventional” immunoglobulins or their fragments, whereintwo immunoglobulin domains, in particular two variable domains, interactto form an antigen binding site. Typically, in conventionalimmunoglobulins, a heavy chain variable domain (VH) and a light chainvariable domain (VL) interact to form an antigen binding site. In thiscase, the complementarity determining regions (CDRs) of both VH and VLwill contribute to the antigen binding site, i.e. a total of 6 CDRs willbe involved in antigen binding site formation.

In contrast, the binding site of an immunoglobulin single variabledomain is formed by a single VH or VL domain. Hence, the antigen bindingsite of an immunoglobulin single variable domain is formed by no morethan three CDRs.

The term “immunoglobulin single variable domain” hence does not compriseconventional immunoglobulins or their fragments which requireinteraction of at least two variable domains for the formation of anantigen binding site. This is also the case for embodiments of theinvention which “comprise” or “contain” an immunoglobulin singlevariable domain. In the context of the present invention, suchembodiments exclude conventional immunoglobulins or their fragments.Thus, a polypeptide or a composition that “comprises” or “contains” animmunoglobulin single variable domain may relate to e.g. constructscomprising more than one immunoglobulin single variable domain.Alternatively, there may be further constituents other than theimmunoglobulin single variable domains, e.g. auxiliary agents ofdifferent kinds, protein tags, colorants, dyes, etc. However, theseterms do comprise fragments of conventional immunoglobulins wherein theantigen binding site is formed by a single variable domain.

Generally, single variable domains will be amino acid sequences thatessentially consist of 4 framework regions (FR1 to FR4 respectively) and3 complementarity determining regions (CDR1 to CDR3 respectively). Suchsingle variable domains and fragments are most preferably such that theycomprise an immunoglobulin fold or are capable for forming, undersuitable conditions, an immunoglobulin fold. As such, the singlevariable domain may for example comprise a light chain variable domainsequence (e.g. a VL-sequence) or a suitable fragment thereof; or a heavychain variable domain sequence (e.g. a VH-sequence or VHH sequence) or asuitable fragment thereof; as long as it is capable of forming a singleantigen binding unit (i.e. a functional antigen binding unit thatessentially consists of the single variable domain, such that the singleantigen binding domain does not need to interact with another variabledomain to form a functional antigen binding unit, as is for example thecase for the variable domains that are present in for exampleconventional antibodies and scFv fragments that need to interact withanother variable domain—e.g. through a VH/VL interaction—to form afunctional antigen binding domain).

In one embodiment of the invention, the immunoglobulin single variabledomains are light chain variable domain sequences (e.g. a VL-sequence),or heavy chain variable domain sequences (e.g. a VH-sequence); morespecifically, the immunoglobulin single variable domains can be heavychain variable domain sequences that are derived from a conventionalfour-chain antibody or heavy chain variable domain sequences that arederived from a heavy chain antibody (e.g. a VHH),

For a general description of heavy chain antibodies and the variabledomains thereof, reference is inter alia made to the prior art citedherein, as well as to the prior art mentioned on page 59 of WO 08/020079and to the list of references mentioned on pages 41-43 of theInternational application WO 06/040153, which prior art and referencesare incorporated herein by reference. As described in these references,Nanobodies (in particular VHH sequences and partially humanizedNanobodies) can in particular be characterized by the presence of one ormore “Hallmark residues” in one or more of the framework sequences. Afurther description of the Nanobodies, including humanization and/orcamelization of Nanobodies, as well as other modifications, parts orfragments, derivatives or “Nanobody fusions”, multivalent constructs(including some non-limiting examples of linker sequences) and differentmodifications to increase the half-life of the Nanobodies and theirpreparations can be found e.g. in WO 08/101985 and WO 08/142164.

For example, the single variable domain or immunoglobulin singlevariable domain (or an amino acid sequence that is suitable for use asan immunoglobulin single variable domain) may be a (single) domainantibody (or an amino acid sequence that is suitable for use as a(single) domain antibody), a “dAb” or dAb (or an amino acid sequencethat is suitable for use as a dAb) or a Nanobody (as defined herein, andincluding but not limited to a VHH sequence); other single variabledomains, or any suitable fragment of any one thereof. For a generaldescription of (single) domain antibodies, reference is also made to theprior art cited herein, as well as to EP 0 368 684. For the term“dAb's”, reference is for example made to Ward et al. 1989 (Nature 341(6242): 544-6), to Holt et al. 2003 (Trends Biotechnol. 21(11):484-490); as well as to for example WO 04/068820, WO 06/030220, WO06/003388 and other published patent applications of Domantis Ltd. Itshould also be noted that, although less preferred in the context of thepresent invention because they are not of mammalian origin, singlevariable domains can be derived from certain species of shark (forexample, the so-called “IgNAR domains”, see for example WO 05/18629).

In particular, the immunoglobulin single variable domain may be aNanobody® (as defined herein) or a suitable fragment thereof. [Note:Nanobody®, Nanobodies® and Nanoclone® are registered trademarks ofAblynx N.V.] For a general description of Nanobodies, reference is madeto the further description below, as well as to the prior art citedherein, such as e.g. described in WO 08/020079 (page 16).

The amino acid sequence and structure of an immunoglobulin sequence, inparticular an immunoglobulin single variable domain can beconsidered—without however being limited thereto—to be comprised of fourframework regions or “FR's”, which are referred to in the art and hereinas “Framework region 1” or “FR1”; as “Framework region 2” or “FR2”; as“Framework region 3” or “FR3”; and as “Framework region 4” or “FR4”,respectively; which framework regions are interrupted by threecomplementary determining regions or “CDR's”, which are referred to inthe art as “Complementarity Determining Region 1” or “CDR1”; as“Complementarity Determining Region 2” or “CDR2”; and as“Complementarity Determining Region 3” or “CDR3”, respectively.

The total number of amino acid residues in an immunoglobulin singlevariable domain can be in the region of 110-120, is preferably 112-115,and is most preferably 113. It should however be noted that parts,fragments, analogs or derivatives of an immunoglobulin single variabledomain are not particularly limited as to their length and/or size, aslong as such parts, fragments, analogs or derivatives meet the furtherrequirements outlined herein and are also preferably suitable for thepurposes described herein.

Thus, in the meaning of the present invention, the term “immunoglobulinsingle variable domain” or “single variable domain” comprises peptideswhich are derived from a non-human source, preferably a camelid,preferably a camel heavy chain antibody. They may be humanized, aspreviously described, e.g. in WO 08/101985 and WO 08/142164. Moreover,the term comprises polypeptides derived from non-camelid sources, e.g.mouse or human, which have been “camelized”, as previously described,e.g. in WO 08/101985 and WO 08/142164.

The term “immunoglobulin single variable domain” encompassesimmunoglobulin sequences of different origin, comprising mouse, rat,rabbit, donkey, human and camelid immunoglobulin sequences. It alsoincludes fully human, humanized or chimeric immunoglobulin sequences.For example, it comprises camelid immunoglobulin sequences and humanizedcamelid immunoglobulin sequences, or camelized immunoglobulin singlevariable domains, e.g. camelized dAb as described by Ward et al (see forexample WO 94/04678 and Davies and Riechmann 1994, Febs Lett. 339: 285and 1996, Protein Engineering 9: 531).

All the ISVDs against vWF (or vWF binders) mentioned above are wellknown from the literature. This includes their manufacture (see inparticular e.g. WO2006/122825 but also WO2004/062551). For example, ALX0081 is prepared as described e.g. in WO2006/122825 or WO2009/115614.

The immunoglobulin single variable domains provided by the invention arepreferably in isolated form or essentially isolated form, or form partof a protein or polypeptide of the invention, which may comprise oressentially consist of one or more immunoglobulin single variabledomains and which may optionally further comprise one or more furtheramino acid sequences (all optionally linked via one or more suitablelinkers). For example, and without limitation, the one or moreimmunoglobulin single variable domains may be used as a binding unit insuch a protein or polypeptide, which may optionally contain one or morefurther amino acid sequences that can serve as a binding unit (i.e.against one or more other targets than cell associated antigens), so asto provide a monovalent, multivalent or multispecific polypeptide of theinvention, respectively, all as described herein. Such a protein orpolypeptide may also be in isolated or essentially isolated form. Thus,according to the invention, immunoglobulin single variable domainscomprise constructs comprising two or more antigen binding units in theform of single domains, as outlined above. For example, two (or more)immunoglobulin single variable domains with the same or differentantigen specificity can be linked to form e.g. a bivalent, trivalent ormultivalent construct. By combining immunoglobulin single variabledomains of two or more specificities, bispecific, trispecific etc.constructs can be formed. For example, a polypeptide according to theinvention may comprise two immunoglobulin single variable domainsdirected against target A, and one immunoglobulin single variable domainagainst target B, making it bivalent for A and monovalent for B. Suchconstructs and modifications thereof, which the skilled person canreadily envisage, are all encompassed by the present invention. Inparticular embodiments, the invention relates to bi-paratopic constructscomprising at least two immunoglobulin single variable domains directedto different epitopes within the same target antigen.

All these molecules are also referred to as “polypeptide of theinvention”, which is synonymous with “immunoglobulin sequences” or“immunoglobulin single variable domains” of the invention.

In addition, the term “sequence” as used herein (for example in termslike “immunoglobulin sequence”, “antibody sequence”, “variable domainsequence”, “V_(HH)-sequence” or “protein sequence”), should generally beunderstood to include both the relevant amino acid sequence as well asnucleic acid sequences or nucleotide sequences encoding the same, unlessthe context requires a more limited interpretation.

According to one non-limiting embodiment of the invention, theimmunoglobulin sequences, Nanobody® or polypeptide of the invention isglycosylated. According to another non-limiting embodiment of theinvention, the immunoglobulin sequences, Nanobody® or polypeptide of theinvention is non-glycosylated.

As mentioned supra, the present invention relates to polypeptidestypically comprising at least one, such as 2 or more ISVDs against vWF,i.e. ISVDs that bind and/or have affinity for an antigen as definedherein, e.g. von Willebrand Factor (vWF) and preferably human vWF (SEQID NO: 20).

In the context of the present invention, “binding to and/or havingaffinity for” a certain antigen has the usual meaning in the art asunderstood e.g. in the context of antibodies and their respectiveantigens.

In particular embodiments of the invention, the term “binds to and/orhaving affinity for” means that the immunoglobulin sequence specificallyinteracts with an antigen, and is used interchangeably withimmunoglobulin sequences “against” the said antigen.

The term “specificity” refers to the number of different types ofantigens or antigenic determinants to which a particular immunoglobulinsequence, antigen-binding molecule or antigen-binding protein (such as aNanobody® or a polypeptide of the invention) can bind. The specificityof an antigen-binding protein can be determined based on affinity and/oravidity. The affinity, represented by the equilibrium constant for thedissociation of an antigen with an antigen-binding protein (KD), is ameasure for the binding strength between an antigenic determinant and anantigen-binding site on the antigen-binding protein: the lesser thevalue of the KD, the stronger the binding strength between an antigenicdeterminant and the antigen-binding molecule (alternatively, theaffinity can also be expressed as the affinity constant (KA), which is1/KD). As will be clear to the skilled person (for example on the basisof the further disclosure herein), affinity can be determined in amanner known per se, depending on the specific antigen of interest.Avidity is the measure of the strength of binding between anantigen-binding molecule (such as a Nanobody® or polypeptide of theinvention) and the pertinent antigen. Avidity is related to both theaffinity between an antigenic determinant and its antigen binding siteon the antigen-binding molecule and the number of pertinent bindingsites present on the antigen-binding molecule.

Typically, immunoglobulin sequences of the present invention (such asthe amino acid sequences, Nanobodies® and/or polypeptides of theinvention) will bind to their antigen with a dissociation constant (KD)of 10⁻⁵ to 10⁻¹² moles/liter or less, and preferably 10⁻⁷ to 10⁻¹²moles/liter or less and more preferably 10⁻⁸ to 10⁻¹² moles/liter (i.e.with an association constant (KA) of 10⁵ to 10¹² liter/moles or more,and preferably 10⁷ to 10¹² liter/moles or more and more preferably 10⁸to 10¹² liter/moles), and/or bind to cell associated antigens as definedherein with a kon-rate of between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹,preferably between 10³ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, more preferably between10⁴ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹, such as between 10⁵ M⁻¹s⁻¹ and 10⁷ M⁻¹s⁻¹;and/or bind to cell associated antigens as defined herein with a koffrate between 1s⁻¹ (t½=0.69 s) and 10⁻⁶ s⁻¹ (providing a nearirreversible complex with a t½ of multiple days), preferably between10⁻² s⁻¹ and 10⁻⁶ s⁻¹, more preferably between 10⁻³ s⁻¹ and 10⁻⁶ s⁻¹,such as between 10⁻⁴ s⁻¹ and 10⁻⁶ s⁻¹.

Any KD value greater than 10⁻⁴ M (or any KA value lower than 10⁴ M⁻¹) isgenerally considered to indicate non-specific binding.

Preferably, a monovalent immunoglobulin sequence of the invention willbind to the desired antigen with an affinity less than 500 nM,preferably less than 200 nM, more preferably less than 10 nM, such asless than 500 pM.

Specific binding of an antigen-binding protein to an antigen orantigenic determinant can be determined in any suitable manner known perse, including, for example, Scatchard analysis and/or competitivebinding assays, such as radioimmunoassays (RIA), enzyme immunoassays(EIA) and sandwich competition assays, and the different variantsthereof known per se in the art; as well as the other techniquesmentioned herein.

The dissociation constant (KD) may be the actual or apparentdissociation constant, as will be clear to the skilled person. Methodsfor determining the dissociation constant will be clear to the skilledperson, and for example include the techniques mentioned herein. In thisrespect, it will also be clear that it may not be possible to measuredissociation constants of more than 10⁻⁴ moles/liter or 10⁻³ moles/liter(e.g., of 10⁻² moles/liter). Optionally, as will also be clear to theskilled person, the (actual or apparent) dissociation constant may becalculated on the basis of the (actual or apparent) association constant(KA), by means of the relationship [KD=1/KA].

The affinity denotes the strength or stability of a molecularinteraction. The affinity is commonly given as by the KD, ordissociation constant, which has units of mol/liter (or M). The affinitycan also be expressed as an association constant, KA, which equals 1/KDand has units of (mol/liter)⁻¹ (or M⁻¹). In the present specification,the stability of the interaction between two molecules (such as an aminoacid sequence, immunoglobulin sequence, Nanobody® or polypeptide of theinvention and its intended target) will mainly be expressed in terms ofthe KD value of their interaction; it being clear to the skilled personthat in view of the relation KA=1/KD, specifying the strength ofmolecular interaction by its KD value can also be used to calculate thecorresponding KA value. The KD-value characterizes the strength of amolecular interaction also in a thermodynamic sense as it is related tothe free energy (DG) of binding by the well-known relation DG=RT.ln(KD)(equivalently DG=−RT.ln(KA)), where R equals the gas constant, T equalsthe absolute temperature and ln denotes the natural logarithm.

The KD for biological interactions, such as the binding of theimmunoglobulin sequences of the invention to the cell associated antigenas defined herein, which are considered meaningful (e.g. specific) aretypically in the range of 10⁻¹⁰ M (0.1 nM) to 10⁻⁵ M (10000 nM). Thestronger an interaction is, the lower is its KD.

The KD can also be expressed as the ratio of the dissociation rateconstant of a complex, denoted as koff, to the rate of its association,denoted kon (so that KD=koff/kon and KA=kon/koff). The off-rate koff hasunits s⁻¹ (where s is the SI unit notation of second). The on-rate konhas units M⁻¹s⁻¹.

As regards immunoglobulin sequences of the invention, the on-rate mayvary between 10² M⁻¹s⁻¹ to about 10⁷ M⁻¹s⁻¹, approaching thediffusion-limited association rate constant for bimolecularinteractions. The off-rate is related to the half-life of a givenmolecular interaction by the relation t½=ln(2)/koff. The off-rate ofimmunoglobulin sequences of the invention may vary between 10⁻⁶ s⁻¹(near irreversible complex with a t½ of multiple days) to 1 s⁻¹ (t½=0.69s).

The affinity of a molecular interaction between two molecules can bemeasured via different techniques known per se, such as the well-knownsurface plasmon resonance (SPR) biosensor technique (see for exampleOber et al., Intern. Immunology, 13, 1551-1559, 2001) where one moleculeis immobilized on the biosensor chip and the other molecule is passedover the immobilized molecule under flow conditions yielding kon, koffmeasurements and hence KD (or KA) values. This can for example beperformed using the well-known Biacore instruments.

It will also be clear to the skilled person that the measured KD maycorrespond to the apparent KD if the measuring process somehowinfluences the intrinsic binding affinity of the implied molecules forexample by artefacts related to the coating on the biosensor of onemolecule. Also, an apparent KD may be measured if one molecule containsmore than one recognition sites for the other molecule. In suchsituation the measured affinity may be affected by the avidity of theinteraction by the two molecules.

Another approach that may be used to assess affinity is the 2-step ELISA(Enzyme-Linked Immunosorbent Assay) procedure of Friguet et al. (J.Immunol. Methods, 77, 305-19, 1985). This method establishes a solutionphase binding equilibrium measurement and avoids possible artefactsrelating to adsorption of one of the molecules on a support such asplastic.

However, the accurate measurement of KD may be quite labour-intensiveand as consequence, often apparent KD values are determined to assessthe binding strength of two molecules. It should be noted that as longas all measurements are made in a consistent way (e.g. keeping the assayconditions unchanged) apparent KD measurements can be used as anapproximation of the true KD and hence in the present document KD andapparent KD should be treated with equal importance or relevance.

Finally, it should be noted that in many situations the experiencedscientist may judge it to be convenient to determine the bindingaffinity relative to some reference molecule. For example, to assess thebinding strength between molecules A and B, one may e.g. use a referencemolecule C that is known to bind to B and that is suitably labelled witha fluorophore or chromophore group or other chemical moiety, such asbiotin for easy detection in an ELISA or FACS (Fluorescent activatedcell sorting) or other format (the fluorophore for fluorescencedetection, the chromophore for light absorption detection, the biotinfor streptavidin-mediated ELISA detection). Typically, the referencemolecule C is kept at a fixed concentration and the concentration of Ais varied for a given concentration or amount of B. As a result an IC50value is obtained corresponding to the concentration of A at which thesignal measured for C in absence of A is halved. Provided KD ref, the KDof the reference molecule, is known, as well as the total concentrationcref of the reference molecule, the apparent KD for the interaction A-Bcan be obtained from following formula: KD=IC50/(1+cref/KD ref). Notethat if cref <<KD ref, KD≈IC50. Provided the measurement of the IC50 isperformed in a consistent way (e.g. keeping cref fixed) for the bindersthat are compared, the strength or stability of a molecular interactioncan be assessed by the IC50 and this measurement is judged as equivalentto KD or to apparent KD throughout this text.

The present invention relates to immunoglobulin single variable domainsdescribed in, or obtainable by the methods as disclosed inWO2004/015425, WO2004/062551, WO2006/074947, WO2006/122825,WO2009/115614, or WO2011/067160, all in the name of the presentapplicant.

The invention also encompasses optimized variants of these amino acidsequences. Generally, an “optimized variant” of an amino acid sequenceaccording to the invention is a variant that comprises one or morebeneficial substitutions such as a substitutions increasing i) thedegree of “humanization”, ii) the chemical stability, and/or iii) thelevel of expression; while the potency (measured e.g. by the potencyassay as described in the experimental part of WO2006/122825 remainscomparable (i.e. within a 10% deviation) to the wild type 12A02 (asdefined in WO2006/122825) or comparable to the variant 12A02H1 (SEQ IDNO: 19), also as defined in WO2006/122825. Preferably, compared to thewild-type sequence of 12A02, an amino acid sequence of the inventioncontains at least one such substitution, and preferably at least twosuch substitutions, and preferably at least three humanizingsubstitutions and preferably at least 10 such humanizing substitutions.

In a particular aspect, the amino acid sequences of the inventioncontain a total of between 1 and 15, preferably between 2 and 14, suchas between 9 and 13, e.g. 10, 11 or 12 amino acid substitutions comparedto the wild-type sequence 12A02. As mentioned, these differencespreferably at least comprise one and preferably at least two, such asthree, four or five or ten humanizing substitutions, and may optionallycomprise one or more further substitutions (such as any one of, or anysuitable combination of any two or more of, the further substitutions(a) to (c) as mentioned herein). Again, based on the disclosure hereinand optionally after a limited degree of trial and error, the skilledperson will be able to select (a suitable combination of) one or moresuch suitable humanizing and/or further substitutions,

The present invention encompasses polypeptide sequences that are highlysimilar to any of the specific examples provided herein, or any of thespecific examples defined by reference above. Highly similar means anamino acid identity of at least 90%, e.g. 95, 97, 98 or 99%. The highlysimilar polypeptide sequences will have the same function as thesequence they are derived from, i.e. they will bind to vWF, morespecifically bind to and inhibit interaction between vWF and platelets.

In a particular embodiment, the invention relates to sequences highlysimilar to any one of SEQ ID NO:s 1-19, in particular SEQ ID NO: 1.However, for each variant sequence stability in the formulation asdefined herein has to be evaluated, such that the invention inparticular refers to variants or highly similar sequences which arestable in the formulations as defined herein.

Methods to generate polypeptide sequences of the invention are widelyknown and include e.g. recombinant expression or synthesis. The skilledperson is well acquainted with suitable expression technology, e.g.suitable recombinant vectors and host cells, e.g. bacterial or yeasthost cells. The skilled person is also well acquainted with suitablepurification techniques and protocols.

The present invention provides also formulations of polypeptidescomprising at least one immunoglobulin single variable domain againstvWF, e.g. ALX 0081, which are stable, and preferably suitable forpharmaceutical uses, including the preparation of medicaments (alsocalled “pharmaceutical formulation of the invention” or “formulation(s)of the invention”).

In particular embodiments, the formulation comprises one or morepolypeptides selected from SEQ ID NO:s 1-19, preferably SEQ ID NO: 1.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient (the polypeptide of the invention) to be effective, and whichcontains no additional components which are unacceptably toxic to asubject to which the formulation would be administered. Suchformulations are sterile, “Pharmaceutically acceptable” excipients(vehicles, additives) are those which can reasonably be administered toa subject mammal to provide an effective dose of the active ingredientemployed,

The term “excipient” as used herein refers to an inert substance whichis commonly used as a diluent, vehicle, preservative, lyoprotectant,surfactant, binder, carrier or stabilizing agent for compounds whichimpart a beneficial physical property to a formulation. The skilledperson is familiar with excipients suitable for pharmaceutical purposes,which may have particular functions in the formulation, such aslyoprotection, stabilization, preservation, etc.

A “sterile” formulation is aseptic or free or essentially free from allliving microorganisms and their spores. This is readily accomplished byfiltration through sterile filtration membranes.

A “stable” formulation is one in which the protein therein essentiallyretains its physical stability and/or chemical stability and/orbiological activity upon storage. Preferably, the formulationessentially retains its physical and chemical stability, as well as itsbiological activity upon storage.

The storage period is generally selected based on the intendedshelf-life of the formulation. Various analytical techniques formeasuring protein stability are available in the art and are reviewed inPeptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., MarcelDekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A, Adv. DrugDelivery Rev. 10: 29-90 (1993), for example. Stability can be measuredat a selected temperature for a selected time period. In certainembodiments, the formulation is stable at about 40° C. for at leastabout 1, 2, 3, 4, 5, 6, 7, 8, or more weeks. Furthermore, theformulation is preferably stable following freezing (to, e.g., −20° C.or −70° C.) and thawing of the formulation, for example following 1, 23, 4, or 5 cycles of freezing and thawing. Stability can be evaluatedqualitatively and/or quantitatively in a variety of different ways knownby the person skilled in the art. Stability studies showed that ALX 0081is stable at −20′C for at least 3 years.

The formulation comprises an aqueous carrier. The aqueous carrier is inparticular a buffer.

As used herein, “buffer” refers to a buffered solution that resistschanges in pH by the action of its acid-base conjugate components. Theformulation of the invention comprises a buffer selected from at leastone of citrate or phosphate buffer, preferably a citrate buffer. Asdetermined previously, these buffers enhance the stability of the vWFbinders.

The formulation according to the invention comprises a citrate buffer ata concentration in the range of 5-200 mM, preferably 7.5-80 mM, evenmore preferably 10-50, e.g. 10, 15, 20, 25 or 30 mM, and most preferably20 mM, wherein each value is understood to optionally encompass a rangeof ±5 mM. Alternatively, the formulation according to the invention maycomprise a phosphate buffer at a concentration in the range of 5-200 mM,preferably 5-80 mM, more preferably 7.5-60 mM, even more preferably10-40, e.g. 10, 15, 20, 25 or 30 mM, and most preferably 10 mM, whereineach value is understood to optionally encompass a range of ±5 mM. Itwill be understood that a lower concentration of the buffer has aneffect on the final osmolality, and correspondingly on the additionalsolutes that may have to be added.

The pH of the formulation of the invention is in the range 5.0 to 7.5,wherein each value is understood to encompass a range of ±0.2. The mostadvantageous pH will depend on the buffer comprised in the formulation.Hence, the invention relates particularly to a formulation comprising aphosphate buffer, which preferably has a pH in the range of 6.5 to 7.5,preferably 6.9, 7.0, 7.1, e.g. 7.1. It was shown that a formulationcomprising a citrate buffer was outstandingly suitable for storage anduse. Hence, the present invention relates to a formulation comprising acitrate buffer, which preferably has a pH between 6.0 and 7.0, morepreferably 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8 or 6.9, e.g. 6.5,wherein each value is understood to optionally encompass a range of±0.2.

The formulations of the invention will comprise the polypeptides of theinvention, in particular the immunoglobulin single variable domains orpolypeptides comprising at least one immunoglobulin single variabledomain against vWF, such as ALX 0081, at a concentration that issuitable for clinical purposes, which includes concentrations used instock solutions for dilution prior to use on the patient. Apart fromimproved stabilization, the formulations of the invention enable highconcentrations of the polypeptides comprising at least one ISVD againstvWF, such as ALX 0081.

Typical concentrations of the active agent, e.g. polypeptides comprisingat least one ISVD against vWF such as ALX 0081, in formulations of theinvention comprise the non-limiting examples of concentrations in therange of 0.1 to 150 mg/mL, such as 1-100 mg/mL, 5-80 mg/mL, or 10-40mg/mL, preferably 10 mg/mL, wherein each value is understood tooptionally encompass a range of ±20% (e.g. a value of 10 optionallyencompasses a range of 8 to 12 mg/mL).

In a further embodiment of the invention, the formulation according toany aspect of the invention may further comprise a detergent orsurfactant.

Herein, a “surfactant” refers to a surface-active agent, preferably anonionic surfactant. Examples of surfactants herein include polysorbate;poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate (SDS);sodium laurel sulfate; sodium octyl glycoside; lauryl-, myristyl-,linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl- orstearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine;lauroamidopropyl-, cocamidopropyl-, linoleamidopropyl-,myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (e.g.lauroamidopropyl); myristamidopropyl-, palmidopropyl-, orisostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or disodiummethyl oleyl-taurate; and the MONAQUAT® series (Mona Industries, Inc.,Paterson, N.J.); polyethyl glycol, polypropyl glycol, and copolymers ofethylene and propylene glycol (e.g. Pluronics, PF68 etc); etc. In oneembodiment, the surfactant herein is polysorbate 80. Preferred suitabledetergents or surfactants for use with the invention include, but arenot limited to, polyoxyethylene sorbitan fatty acid esters e.g.polysorbate 20, −40, −60, −65, −80 or −85. Common brand names forpolysorbates include Alkest, Canarcel and Tween. The skilled personknows further non-limiting examples of detergents, such as those listede.g. in WO2010/077422. In a preferred embodiment, the detergent is anon-ionic detergent. More specifically, the detergent is polysorbate-80,also designated Tween-80 hereafter. The skilled person can readilydetermine a suitable concentration of detergent for a formulation of theinvention. Typically, the concentration will be as low as possible,whilst maintaining the beneficial effects of the detergents, e.g. astabilizing effect under conditions of shear stress, e.g. stirring,which reduces aggregation of the formulated polypeptides of theinvention. In exemplary, non-limiting embodiments, the concentration ofthe detergent may be in the range of 0.001 to 0.5%, e.g. 0.001, 0.002,0.003, 0.004, 0.005, 0.01, 0.015, 0.02, 0.025, 0.03, 0.035, 0.04, 0.045,0.05%, 0.1%, 0.2%, 0.3%, 0.4% or 0.5%, preferably in a concentrationbetween 0.01 and 0.05%, more preferably between 0.01 and 0.02%, e.g.0.01% (v/v).

The formulation of the invention may further comprise excipients such aspreservatives.

A “preservative” is a compound which can be optionally included in theformulation to essentially reduce bacterial action therein, thusfacilitating the production of a multi-use formulation, for example.Examples of potential preservatives include octadecyldimethylbenzylammonium chloride, hexamethonium chloride, benzalkonium chloride (amixture of alkylbenzyldimethylammonium chlorides in which the alkylgroups are long-chain compounds), and benzethonium chloride. Other typesof preservatives include aromatic alcohols such as phenol, butyl andbenzyl alcohol, alkyl parabens such as methyl or propyl paraben,catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol. In oneembodiment, the preservative herein is benzyl alcohol.

The formulation of the invention may further comprise stabilizingagents, such as a polyols.

A “polyol” is a substance with multiple hydroxyl groups, and includessugars (reducing and nonreducing sugars), sugar alcohols and sugaracids. A polyol may optionally be included in the formulation, forinstance to improve stability. In certain embodiments, polyols hereinhave a molecular weight which is less than about 600 kD (e.g. in therange from about 120 to about 400 kD). A “reducing sugar” is one whichcontains a hemi-acetal group that can reduce metal ions or reactcovalently with lysine and other amino groups in proteins and a“nonreducing sugar” is one which does not have these properties of areducing sugar. Examples of reducing sugars are fructose, mannose,maltose, lactose, arabinose, xylose, ribose, rhamnose, galactose andglucose. Nonreducing sugars include sucrose, trehalose, sorbose,melezitose and raffinose. Mannitol, xylitol, erythritol, threitol,sorbitol and glycerol are examples of sugar alcohols. As to sugar acids,these include L-gluconate and metallic salts thereof. Where it desiredthat the formulation is freeze-thaw stable, the polyol is preferably onewhich does not crystallize at freezing temperatures (e.g. −20° C.) suchthat it destabilizes the antibody in the formulation. In certainembodiments, nonreducing sugars such as sucrose and trehalose areexamples of polyols, with sucrose being preferred, despite the solutionstability of trehalose.

Therapeutic compounds of the invention used in accordance with thepresent invention are prepared for storage by mixing a polypeptide(s)having the desired degree of purity with optional pharmaceuticallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the formof lyophilized formulations or aqueous solutions. Acceptable carriers,excipients, or stabilizers are nontoxic to recipients at the dosages andconcentrations employed. Accordingly, the formulations according to theinvention may also optionally comprise one or more excipients.

Commonly used stabilizers and preservatives are well known to theskilled person (see e.g. WO2010/077422). Pharmaceutically acceptablecarriers that may be used in these compositions include, but are notlimited to, ion exchangers, alumina, aluminum stearate, lecithin, serumproteins, such as human serum albumin, buffer substances such asphosphates, glycine, sorbic acid, potassium sorbate, partial glyceridemixtures of saturated vegetable fatty acids, water, salts orelectrolytes, such as protamine sulfate, disodium hydrogen phosphate,potassium hydrogen phosphate, sodium chloride, zinc salts, colloidalsilica, magnesium trisilicate, hydrophilic polymers such as polyvinylpyrrolidone, cellulose based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes, gelatin, polyethylenepolyoxypropylene block polymers, polyethylene glycol and wool fat.antioxidants including ascorbic acid and methionine; preservatives; lowmolecular weight (less than about 10 residues) polypeptides; proteins;and amino acids such as glycine, glutamine, asparagine, histidine,arginine, or lysine. In advantageous embodiments, the excipient may beone or more selected from the list consisting of NaCl, trehalose,sucrose, mannitol or glycine.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in microemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The polypeptides of the invention may be formulated into anypharmaceutically acceptable formulation. The formulation may be liquidor dry. The formulation may be generated via mixing, drying,lyophilization, vacuum drying, or any known method for formulatingpharmaceutical compositions.

A preferred formulation of the invention comprises a polypeptidecomprising at least one ISVD against vWF, such as ALX 0081, in aphosphate buffer solution (pH 7.1). Even more preferably, a formulationof the invention comprises a polypeptide comprising at least one ISVDagainst vWF, such as ALX 0081, in a phosphate buffer solution (pH 7.1),Glycine (0.2 M) and polysorbate 80 (0.02% v/v).

The polypeptides of the invention may further be formulated as describedin PCT/EP14/060107.

A preferred formulation comprises:

-   (a) a polypeptide comprising at least one ISVD against vWF, such as    ALX 0081 at a concentration from about 0.1 mg/mL to about 80 mg/mL;-   (b) an excipient chosen from sucrose, glycine, mannitol, trehalose    or NaCl at a concentration of about 1% to about 15% (w/v);-   (c) Tween-80 at a concentration of about 0.001% to 0.5% (v/v); and-   (d) a citrate buffer at a concentration of about 5 mM to about 200    mM such that the pH of the formulation is about 6.0 to 7.0.

A further preferred formulation of the invention comprises a polypeptidecomprising at least one ISVD against vWF, such as ALX 0081, preferablyat a concentration of 10 mg/ml, a citrate buffer at a concentration of20 mM (pH 6.5), further comprising 7% sucrose (w/v), and Tween-80 at aconcentration of 0.01% (v/v).

In some embodiments, a formulation is stored as a liquid. In otherembodiments, a formulation is prepared as a liquid and then is dried,e.g., by lyophilization or spray-drying, prior to storage. A driedformulation can be used as a dry compound, e.g., as an aerosol orpowder, or reconstituted to its original or another concentration, e.g.,using water, a buffer, or other appropriate liquid.

The present invention also relates to vials comprising filled withlyophilisate containing 12.5 mg caplacizumab and excipients for solutionfor injection. Excipients (per mL of reconstituted solution): 0.21 mgcitric acid, 5.58 mg tri sodium citrate di-hydrate, 70 mg sucrose, 0.11mg polysorbate-80 per vial (pH 6.5+/−0.5). After reconstitution with 1mL Water for injection (WFI) strength is 12.5 mg/mL caplacizumab (foradministered nominal dose of 10 mg).

The invention also encompasses products obtainable by further processingof a liquid formulation, such as a frozen, lyophilized or spray-driedproduct. Upon reconstitution, these solid products can become liquidformulations as described herein (but are not limited thereto). In itsbroadest sense, therefore, the term “formulation” encompasses bothliquid and solid formulations. However, solid formulations areunderstood as derivable from the liquid formulations (e.g. by freezing,freeze-drying or spray-drying), and hence have various characteristicsthat are defined by the features specified for liquid formulationsherein. The invention does not exclude reconstitution that leads to acomposition that deviates from the original composition before e.g.freeze- or spray drying. accordingly, the lyophilized formulation may bereconstituted to produce a formulation that has a concentration thatdiffers from the original concentration (i.e., before lyophilization),depending upon the amount of water or diluent added to the lyophilaterelative to the volume of liquid that was originally freeze-dried.Suitable formulations can be identified by assaying one or moreparameters of antibody integrity.

In a preferred embodiment, the formulations according to the inventionare isotonic in relation to human blood. Isotonic solutions possess thesame osmotic pressure as blood plasma, and so can be intravenouslyinfused into a subject without changing the osmotic pressure of thesubject's blood plasma. Tonicity can be expressed in terms ofosmolality, which can be a theoretical osmolality, or preferably anexperimentally determined osmolality. Typically, osmolality will be inthe range of 290±60 mOsm/kg, preferably 290±20 mOsm/kg.

The formulations of the invention may also comprise compounds that arespecifically useful for protecting the polypeptide of the inventionduring freeze-drying. Such compounds are also known as lyoprotectants,and are well known to the skilled person. Specific examples include, butare not limited to sugars like sucrose, sorbitol or trehalose; aminoacids such as glutamate, in particular monosodium glutamate orhistidine; betain, magnesium sulfate, sugar alcohols, propylene glycol,polyethylene glycols and combinations thereof. By appreciating theinvention, the required amount of such a compound to be added canreadily be determined by the skilled person under consideration ofstability of the formulation in liquid form and when undergoinglyophilization. Formulations that are particularly suitable forfreeze-drying may furthermore comprise bulking agents. Suitable agentsare widely known to the skilled person. It has been shown that aformulation comprising sucrose was not only particularly suited formaintaining the physical stability, during e.g. storage andfreeze-thawing, of the vWF binders, but also as lyoprotectant.

As outlined, any of the above formulations can be further processed e.g.by lyophilization, spray-drying or freezing, e.g. bulk freezing. Theresulting processed product has characteristics derived from the liquidstarting formulation, as defined above. Where necessary, additionalagents may be included for further processing, such as, for instance,lyoprotectants, etc.

The formulations of the present invention have the effect afterlyophilization of maintaining the chemical and physical integrity of thepolypeptides of the present invention, in particular ALX 0081, i.e. evenafter prolonged storage, e.g. for durations as defined above, attemperatures between −70° C. and +40° C., the purity/impurity profile ofthe product is essentially not changing. For example, prolonged storageafter lyophilization did not have a significant effect on RP-HPLC,SE-HPLC or clEF profiles.

The polypeptides of the invention can be produced by any commonly usedmethod. Typical examples include the recombinant expression in suitablehost systems, e.g. bacteria or yeast. The polypeptides of the inventionwill undergo a suitable purification regimen prior to being formulatedin accordance to the present invention.

In general, the polypeptides of the invention are produced by livinghost cells that have been genetically engineered to produce thepolypeptide. Methods of genetically engineering cells to produceproteins are well known in the art. See e.g. Ausubel et al., eds.(1990), Current Protocols in Molecular Biology (Wiley, New York). Suchmethods include introducing nucleic acids that encode and allowexpression of the polypeptide into living host cells. These host cellscan be bacterial cells, fungal cells, or animal cells grown in culture.Bacterial host cells include, but are not limited to, Escherichia colicells. Examples of suitable E. coli strains include: HB101, DH5a,GM2929, JM109, KW251, NM538, NM539, and any E. coli strain that fails tocleave foreign DNA. Fungal host cells that can be used include, but arenot limited to, Saccharomyces cerevisiae, Pichia pastoris andAspergillus cells. A few examples of animal cell lines that can be usedare CHO, VERO, BHK, HeLa, Cos, MDCK, 293, 3T3, and Wl38. New animal celllines can be established using methods well known by those skilled inthe art (e.g., by transformation, viral infection, and/or selection).Optionally, the polypeptide can be secreted by the host cells into themedium.

In some embodiments, the polypeptides can be produced in bacterialcells, e.g., E. coli cells. For example, if the polypeptide is encodedby sequences in a phage display vector that includes a suppressible stopcodon between the display entity and a bacteriophage protein (orfragment thereof), the vector nucleic acid can be transferred into abacterial cell that cannot suppress a stop codon. In this case, thepolypeptide is not fused to the gene III protein and is secreted intothe periplasm and/or media.

The polypeptides can also be produced in eukaryotic cells. In oneembodiment, the polypeptides are expressed in a yeast cell such asPichia (see, e.g., Powers et al. J Immunol Methods 251:123-35 (2001)),Hansenula, or Saccharomyces.

In one embodiment, polypeptides are produced in mammalian cells. Typicalmammalian host cells for expressing the clone antibodies orantigen-binding fragments thereof include Chinese Hamster Ovary (CHOcells) (including dhfr—CHO cells, described in Urlaub and Chasin, Proc.Natl. Acad. Sci. USA 77:4216-4220(1980), used with a DHFR selectablemarker, e.g., as described in Kaufman and Sharp, Mol. Biol. 159:601-621(1982)), lymphocytic cell lines, e.g., NSO myeloma cells and SP2 cells,COS cells, and a cell from a transgenic animal, e.g., a transgenicmammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequences encoding the polypeptide, therecombinant expression vectors may carry additional sequences, such assequences that regulate replication of the vector in host cells (e.g.,origins of replication) and selectable marker genes. The selectablemarker gene facilitates selection of host cells into which the vectorhas been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and5,179,017). For example, typically the selectable marker gene confersresistance to drugs, such as G418, hygromycin, or methotrexate, on ahost cell into which the vector has been introduced.

Standard molecular biology techniques can be used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody moleculefrom the culture medium. For example, the polypeptides of the inventioncan be isolated by affinity chromatography.

In one embodiment, the polypeptide of the invention is purified asdescribed in WO 10/056550. In an exemplary embodiment, the polypeptideis purified from one or more contaminants by: contacting a mixture ofpolypeptide and contaminant(s) with a Protein A-based support and/or anion exchange support, under conditions that allow the polypeptide tobind to or adsorb to the support; removing one or more contaminants bywashing the bound support under conditions where the polypeptide remainsbound to the support, and selectively eluting the polypeptide from thesupport by eluting the adsorbed polypeptide molecule with an elutionbuffer.

The polypeptides of the invention can also be produced by a transgenicanimal. For example, U.S. Pat. No. 5,849,992 describes a method ofexpressing an antibody in the mammary gland of a transgenic mammal. Atransgene is constructed that includes a milk-specific promoter andnucleic acids encoding the antibody molecule and a signal sequence forsecretion. The milk produced by females of such transgenic mammalsincludes, secreted therein, the single domain of interest. The antibodymolecule can be purified from the milk, or for some applications, useddirectly.

The present invention encompasses methods of producing the formulationsas defined herein.

The purification and formulation steps may coincide, e.g. when thepolypeptides of the invention are eluted from a column using a bufferaccording to the present invention. Alternatively, the formulations ofthe invention can be prepared by exchanging a buffer by any suitablemeans, e.g. means widely used in the art such as dialyzing,ultrafiltration, etc.

In some embodiments the method of producing a formulation of theinvention may also relate to the reconstitution of a lyophilized orspray-dried formulation, e.g. by addition of water or a suitable buffer(which may optionally comprise further excipients).

The methods for preparing a formulation according to the presentinvention may encompass further steps, such as filling it into vialssuitable for clinical use, such as sealed containers and/orconfectioning it in a dosage unit form. The methods may also comprisefurther steps such as spray-drying, lyophilization, or freezing, e.g.bulk freezing. The invention also encompasses the containers, dosageunit forms, or other products obtainable by any of the methods recitedherein.

The formulations of the present invention can be used to store thepolypeptides of the invention, e.g. polypeptides comprising at least oneISVD against vWF, such as ALX 0081, as defined herein. Thus, theinvention encompasses a method of storage of the polypeptides of theinvention as used herein, characterized by the use of a formulation asdefined herein. More specifically, the invention encompasses methods forstabilizing the polypeptides of the invention for storage, comprisinge.g. the preparation of a formulation as described herein. Storage canbe 1-36 months, such as 1, 1.5, 3, 6, 9, 12, 18, 24, 30 or 36 months,e.g. at least 12 months, optionally at a temperature between −70° C. and+40° C., such as −70′C, −20° C., +5° C., +25° C. or +40° C., preferablya temperature between −70° C. and +25° C., more preferably at atemperature between −20° C. and +5° C. Thus, storage may encompassfreezing, freeze-drying (lyophilization) and/or spray-drying. Thestorage methods may furthermore comprise the assessment of physical andchemical integrity of the vWF binders as defined herein,

The present invention also relates to methods for analyzing formulationscomprising at least one of the vWF binders as defined herein. Theformulations can be analyzed for any signs of chemical or physicalinstability of the vWF binders as defined herein. For example, theformulations can be assessed for the presence of degradation products,e.g. low molecular weight derivatives such as proteolytic fragments;and/or for chemical derivatives, e.g. pyroglutamate variants; and/or forhigh molecular weight derivatives such as aggregates, agglomerates, etc.The formulation can also be assessed for total protein content and/orpotency. Each of the various assay methods as referred to herein can beused in the analysis method of the present invention.

Thus, the present invention also relates to a method for monitoringand/or assessing the quality and/or stability of a formulation, e.g.during one or more of manufacture, storage and use. The invention alsorelates to a method of quality control of a formulation, e.g. to assessthat the formulation meets product specifications as further describedherein. The invention in any of these aspects comprises one or moreselected from the comparison with one or more reference samples, theanalysis of batch to batch variation, and the ongoing monitoring of aproduction process.

The present invention relates to any product that is associated with theformulations of the present invention, e.g. by comprising them, or bybeing necessary for their production or confectioning, without anylimitations.

For example, the present invention relates to an article of manufacture,e.g. a sealed container comprising one or more of the formulationsaccording to the present invention.

The invention also relates to a pharmaceutical unit dosage form, e.g. adosage form suitable for parenteral administration (e.g., intradermally,intramuscularly, intraperitoneally, intravenously and subcutaneously) toa patient, preferably a human patient, comprising one or more of theformulation according to any embodiment described herein.

The dosage unit form can be e.g. in the format of a prefilled syringe,an ampoule, cartridge or a vial.

Also provided are kits or articles of manufacture, comprising theformulation of the invention and instructions for use by, e.g., ahealthcare professional. The kits or articles of manufacture may includea vial or a syringe containing the formulation of the invention asdescribed herein.

Preferably, the vial or syringe is composed of glass, plastic, or apolymeric material chosen from a cyclic olefin polymer or copolymer. Thesyringe, ampoule, cartridge or vial can be manufactured of any suitablematerial, such as glass or plastic and may include rubber materials,such as rubber stoppers for vials and rubber plungers and rubber sealsfor syringes and cartridges. The invention also relates to a kitcomprising one or more of the formulations according to the presentinvention. The kit may further comprise instructions for use and/or aclinical package leaflet. In any embodiment of the products as definedherein, the invention also encompasses the presence of packagingmaterial, instructions for use, and/or clinical package leaflets, e.g.as required by regulatory aspects.

For the purposes of comparing two or more amino acid sequences, thepercentage of “sequence identity” between a first amino acid sequenceand a second amino acid sequence (also referred to herein as “amino acididentity”) may be calculated by dividing [the number of amino acidresidues in the first amino acid sequence that are identical to theamino acid residues at the corresponding positions in the second aminoacid sequence] by [the total number of amino acid residues in the firstamino acid sequence] and multiplying by [100%], in which each deletion,insertion, substitution or addition of an amino acid residue in thesecond amino acid sequence—compared to the first amino acid sequence—isconsidered as a difference at a single amino acid residue (position),i.e. as an “amino acid difference” as defined herein.

Alternatively, the degree of sequence identity between two amino acidsequences may be calculated using a known computer algorithm, such asthose mentioned above for determining the degree of sequence identityfor nucleotide sequences, again using standard settings.

Usually, for the purpose of determining the percentage of “sequenceidentity” between two amino acid sequences in accordance with thecalculation method outlined hereinabove, the amino acid sequence withthe greatest number of amino acid residues will be taken as the “first”amino acid sequence, and the other amino acid sequence will be taken asthe “second” amino acid sequence.

Also, in determining the degree of sequence identity between two aminoacid sequences, the skilled person may take into account so-called“conservative” amino acid substitutions, which can generally bedescribed as amino acid substitutions in which an amino acid residue isreplaced with another amino acid residue of similar chemical structureand which has little or essentially no influence on the function,activity or other biological properties of the polypeptide. Suchconservative amino acid substitutions are well known in the art, forexample from WO 04/037999, GB-A-3 357 768, WO 98/49185, WO 00/46383 andWO 01/09300; and (preferred) types and/or combinations of suchsubstitutions may be selected on the basis of the pertinent teachingsfrom WO 04/037999 as well as WO 98/49185 and from the further referencescited therein. Such conservative substitutions preferably aresubstitutions in which one amino acid within the following groups(a)-(e) is substituted by another amino acid residue within the samegroup: (a) small aliphatic, nonpolar or slightly polar residues: Ala,Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their(uncharged) amides: Asp, Asn, Glu and Gln; (c) polar, positively chargedresidues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met,Leu, Ile, Val and Cys; and (e) aromatic residues: Phe, Tyr and Trp.Particularly preferred conservative substitutions are as follows: Alainto Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp intoGlu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro;His into Asn or into Gin; Ile into Leu or into Val; Leu into Ile or intoVal; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or intolie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trpinto Tyr; Tyr into Trp; and/or Phe into Val, into lie or into Leu. Anyamino acid substitutions applied to the polypeptides described hereinmay also be based on the analysis of the frequencies of amino acidvariations between homologous proteins of different species developed bySchulz et al., Principles of Protein Structure, Springer-Verlag, 1978,on the analyses of structure forming potentials developed by Chou andFasman, Biochemistry 13: 211, 1974 and Adv. Enzymol., 47: 45-149, 1978,and on the analysis of hydrophobicity patterns in proteins developed byEisenberg et al., Proc. Natl. Acad. Sci. USA 81: 140-144, 1984; Kyte &Doolittle; J Molec. Biol. 157: 105-132, 1981, and Goldman et al., Ann.Rev. Biophys. Chem. 15: 321-353, 1986, all incorporated herein in theirentirety by reference. Information on the primary, secondary andtertiary structure of Nanobodies® is given in the description herein andin the general background art cited above. Also, for this purpose, thecrystal structure of a V_(HH) domain from a llama is for example givenby Desmyter et al., Nature Structural Biology, Vol. 3, 9, 803 (1996);Spinelli et al., Natural Structural Biology (1996); 3, 752-757; andDecanniere et al., Structure, Vol. 7, 4, 361 (1999). Further informationabout some of the amino acid residues that in conventional V_(H) domainsform the V_(H)/V_(L) interface and potential camelizing substitutions onthese positions can be found in the prior art cited above.

The present invention also relates to a method of treating or preventinga vWF-related disease, such as e.g. acute coronary syndrome (ACS),transient cerebral ischemic attack, unstable or stable angina pectoris,stroke, myocardial infarction or thrombotic thrombocytopenic purpura(TTP); said method comprising administering to a subject apharmaceutical composition comprising the formulation of the invention,thereby reducing one or more symptoms associated with said vWF-relateddisease. In particular, said vWF-related disease is TTP.

In addition, the present invention relates to a method for the treatmentof a human patient susceptible to or diagnosed with a diseasecharacterized by a vWF-related disease, comprising administering aneffective amount of a polypeptide comprising at least one immunoglobulinsingle variable domain (ISVD) against von Willebrand Factor (vWF) to thehuman patient.

The present invention provides a method of treating or preventing avWF-related disease, such as TTP, comprising administering to a human,5-40 mg dose of a polypeptide comprising at least one immunoglobulinsingle variable domain (ISVD) against von Willebrand Factor (vWF),thereby reducing one or more symptoms associated with the vWF-relateddisease.

The present invention provides a treatment as described herein, whereinsaid administering a polypeptide as described herein is followed within5 min to 8 h by performing a first Plasma Exchange (PE).

The present invention provides a treatment as described herein, whereinsaid administering of a polypeptide as described herein is preceded byperforming a preceded Plasma Exchange (PE), within 36 h, preferably 32,30, 28, 26, 24, 22, 20, 18, or 16 h, preferably about 24 h of said firstPE.

The present invention provides a treatment as described herein, whereinsaid first PE is followed by administering a second dose of 1-40 mg,preferably 10 mg of a polypeptide as described herein within 5 min to 8h, such as within 10 min to 6 h or 15 min to 4 h, for instance within 8h, 7 h, 6 h, 5 h, 4 h, 3 h, 3 h, 1 h, 45 min, 30 min, 20 min, 15 min, 10min or even 5 min, for instance wherein said second dose of saidpolypeptide is administered within 1-60 min, such as 30 min of saidfirst PE, preferably by subcutaneous injection.

The present invention provides a treatment as described herein, furthercomprising:

-   (i) performing a PE; (followed by)-   (ii) administering a dose of 5-40 mg of a polypeptide as described    herein 15 min to 4 h after said PE of step (i); and-   (iii) optionally measuring the platelet count and/or ADAMTS13    activity of said patient, wherein step (i) and step (ii) are    repeated once per day until the platelet count of said patient    is >150000/μl and/or the ADAMTS13 activity is at least 10% such as    at least 15%, 20%, 25%, 30%, 35%, 45% or even 50% of the ADAMTS13    reference activity.

The present invention provides also a treatment as described herein,further comprising administering once per day a dose of 5-40 mg of apolypeptide as described herein for at least 5, 10, 15, 20, 25, or even30 days after the platelet count of said patient is >150.000/μl.

The present invention provides a treatment as described herein, furthercomprising administering once per day a dose of 5-40 mg of a polypeptideas described herein until said human enters remission.

The present invention provides a treatment as described herein,comprising administering said polypeptide until the ADAMTS13 activity isat least 10% such as at least 15%, 20%, 25%, 30%, 35%, 45% or even 50%of the ADAMTS13 reference activity.

In another embodiment of the invention, an article of manufacturecontaining materials useful for the treatment of a disease as describedabove is provided. The article of manufacture comprises a contained, alabel and a package insert. Suitable containers include, for example,bottles, vials, syringes, etc. The containers may be of a variety ofmaterials such as glass or plastic. The container holds the compositionwhich is effective in treating the condition and may have a sterileaccess port (for example the container may be an intravenous solutionbag or a vial having a stopper pierceable by a hypodermic injectionneedle). At least one active agent in the composition is the polypeptideof the invention, such as ALX 0081. The label on, or associated with,the container indicates that the composition is used for treating thecondition of choice. The article of manufacture may further comprise asecond container comprising a pharmaceutically acceptable buffer, suchas a phosphate buffer saline or a citrate buffered saline as describedherein. It may further include other materials desirable from a user orcommercial standpoint, including other buffers, diluents, filters,needles and syringes.

The present invention provides a kit or an article of manufacture,comprising a container containing the polypeptide as described herein orthe formulation as described herein, and instructions for use.

The present invention provides a kit or article of manufacture asdescribed herein, wherein the formulation is present in a vial or aninjectable syringe.

The present invention provides a kit or article of manufacture asdescribed herein, wherein the formulation is present in a prefilledinjectable syringe.

The present invention provides a kit or article of manufacture asdescribed herein, wherein the syringe or a vial is composed of glass,plastic, or a polymeric material chosen from a cyclic olefin polymer orcopolymer.

The embodiments illustrated and discussed in this specification areintended only to teach those skilled in the art the best way known tothe inventors to make and use the invention. Modifications and variationof the above-described embodiments of the invention are possible withoutdeparting from the invention, as appreciated by those skilled in the artin light of the above teachings. It is therefore understood that, withinthe scope of the claims and their equivalents, the invention may bepracticed otherwise than as specifically described.

The invention will now be further described by means of the followingnon-limiting preferred aspects, examples and figures.

The entire contents of all of the references (including literaturereferences, issued patents, published patent applications, andco-pending patent applications) cited throughout this application arehereby expressly incorporated by reference, in particular for theteaching that is referenced hereinabove.

6. ABBREVIATIONS

AE adverse eventsACS acute coronary syndromeADAMTS13 a disintegrin-like and metalloprotease with thrombospondinrepeats 13

ALX 0081 Caplacizumab

AMI Acute myocardial infarctionBNP brain natriuretic peptideBMI body mass index

BU Bethesda Units

CDR complementarity determining regionclEF Capillary IsoElectric FocusingdAb single domain antibodyELISA enzyme-linked immunosorbent assay

HR Hazard Ratio

ISVD Immunoglobulin single variable domainITT intent-to-treati.v. intravenousFR framework regionKA association constantKD dissociation constantLDH Lactate dehydrogenaseNSE neuron specific enolaseNT proBNP N-terminal pro brain natriuretic peptidePE or PEX plasma exchangePP per protocolRICO Ristocetin cofactor activity

RP-HPLC Reverse Phase High Performance Liquid Chromatography

SAE serious adverse events.c. subcutaneousscFv single chain variable fragment

S/D Solvent/Detergent SE-HPLC Size Exclusion High Performance LiquidChromatography

SPR surface plasmon resonanceTnI troponin ITnT troponin TTRALI Transfusion related acute lung injuryTTP Thrombotic thrombocytopenic purpuraTTR time-to-responseULN Upper limit normalULvWF ultra-large vWFVH heavy chain variable domainVHH heavy chain variable domain sequence that is derived from a heavychain antibodyVL light chain variable domain

vWF von Willebrand Factor 7. EXAMPLES 7.1 Applicable Regulations

All human samples used in the Examples section were either obtained fromcommercial sources or from human volunteers (after all required consentsand approvals were obtained) and were used in according with theapplicable legal and regulatory requirements (including those regardingmedical secret and patient privacy).

Clinical trials were performed in accordance with applicable laws andregulations (including the Declaration of Helsinki and the principles ofmedical secret and the protection of patient privacy) and after allrequired approvals (including approvals by relevant ethics committees)and consents (including informed consent of subjects involved) wereobtained.

The objectives and contents of this clinical study as well as itsresults were treated as confidential and have not been made accessibleto third parties. Employees participating in the study were bound byconfidentiality. All unused drugs were either returned to the applicantor destroyed.

7.2 Effects of ALX 0081 on Platelet Adhesion to Endothelial Cell-DerivedULvWF and on the Activity of ADAMTS13

Goal: the goal of this study was to evaluate if a polypeptide of theinvention, such as ALX 0081 can inhibit adhesion of platelets to ULvWF.This could then serve as proof of concept for the use of a polypeptideof the invention, such as ALX-081, for treatment of TTP patients in anacute episode. The study also determines the effect of a polypeptide ofthe invention, such as ALX 0081, on the activity of ADAMTS13.

Method: the flow chamber test was used to test whether a polypeptide ofthe invention, such as ALX 0081, can inhibit the interaction betweenplatelets and ULvWF according to Sixma (Sixma et al. 1998 Thromb Res 92:S43-546). In short, endothelial cells were cultivated on coverslips andstimulated, thereby secreting ULvWF. Plasma from TTP patients wassupplemented with platelets and perfused over the stimulated cells.Strings of platelets adhering to ULvWF were visualized by real timevideo-microscopy. The experiment was repeated in the presence ofincreasing concentrations of a polypeptide of the invention, such as ALX0081. In order to determine the effect of a polypeptide of theinvention, such as ALX 0081, on the cleavage of ULvWF by ADAMTS13, twotypes of experiments were used. In the first experiment, cleavage ofplatelet strings by ADAMTS13 was evaluated in the absence of excess apolypeptide of the invention, such as ALX 0081. In the secondexperiment, a recombinant fragment composed of the A1-A2-A3 domain ofvWF was used to evaluate the inhibitory effect of a polypeptide of theinvention, such as ALX 0081, on ADAMTS13.

Results: ALX 0081 inhibited platelet string formation on ULvWF at allconcentrations tested and had no effect on platelet string detachment byADAMTS13. The polypeptide of the invention, such as ALX 0081, also hadno effect on the cleavage of the recombinant A1-A2-A3 domain fragment ofvWF by ADAMTS13. The polypeptide of the invention, such as ALX 0081, wasalso not able to dislodge the platelets from already formed strings inthis experiment.

Conclusion: this study delivers a proof of concept that a polypeptide ofthe invention, such as ALX 0081, can be used to treat TTP patients. Italso proves that a polypeptide of the invention, such as ALX 0081, doesnot interfere with the ADAMTS13 activity.

7.3 Eligibility Criteria

Patients had to fulfill all of the following criteria to be eligible forstudy admission:

Inclusion Criteria

-   -   1. 18 years of age or older    -   2. Men or women willing to accept an acceptable contraceptive        regimen    -   3. Patients with clinical diagnosis of TTP    -   4. Necessitating PE (one, single PE session prior to        randomisation into the study is allowed)    -   5. Subject accessible to follow-up    -   6. Obtained, signed and dated informed consent

Exclusion Criteria

-   -   1. Platelet count greater or equal to 100,000/μL    -   2. Severe active infection indicated by sepsis (requirement for        pressors with or without positive blood cultures)    -   3. Clinical evidence of enteric infection with E. coli 0157 or        related organism    -   4. Anti-phospholipid syndrome    -   5. Diagnosis of DIC    -   6. Pregnancy or breast-feeding    -   7. Haematopoietic stem cell or bone marrow        transplantation-associated thrombotic microangiopathy    -   8. Known congenital TTP    -   9. Active bleeding or high risk of bleeding    -   10. Uncontrolled arterial hypertension    -   11. Known chronic treatment with anticoagulant treatment that        can not be stopped safely, including but not limited to vitamin        K antagonists, heparin or LMWH, and non-acetyl salicylic acid        non-steroidal anti-inflammatory molecules    -   12. Severe or life threatening clinical condition other than TTP        that would impair participation in the trial    -   13. Subjects with malignancies resulting in a life expectation        of less than 3 months    -   14. Subjects with known or suspected bone marrow carcinosis    -   15. Subjects who cannot comply with study protocol requirements        and procedures.    -   16. Known hypersensitivity to the active substance or to        excipients of the study drug    -   17. Severe liver impairment, corresponding to grade 3 toxicity        defined by the CTCAE scale. For the key liver parameters, this        is defined as follows:        -   bilirubin >3×ULN (need to differentiate isolated increase in            indirect bilirubin due to haemolysis, this is not an            exclusion parameter but disease related)        -   ALT/AST >5×ULN        -   AP >5×ULN        -   gamma glutamyl transpeptidase (GGT) >5×ULN    -   18. Severe chronic renal impairment, as defined by GFR <30        mL/min

7.4 Study Design

The present study was designed as a Phase II multicentre,single-blinded, parallel design, randomised, placebo-controlled study(Titan trial). The study population were symptomatic patients with acuteepisodes of acquired TTP, requiring treatment with PE. Afterconfirmation of eligibility to study participation (cf. Example 7.3),patients were randomised in a ratio of 1:1 to either receive ALX 0081 orplacebo as adjunctive therapy to PE (FIG. 1). Patients were randomisedprior to the start of PE treatment. In exceptional cases however (due toneed or ability to start PE in a time frame which did not allow allrequired screening and/or baseline study procedures to be performed), apatient was randomised after a preceding, single PE session (“precedingPE”), but prior to the start of the next PE session (“first PE”). Thisoverall next PE session was started within 24 hours of the end of thepreceding PE session, and was considered the first PE-on-study (“firstPE”).

The patients were followed in different phases during this study:

-   -   Screening and baseline measurements after admission to hospital    -   Treatment phase        -   Single i.v. bolus study drug administered via push injection        -   Daily PE adjunctive s.c. treatment phase        -   Post-daily PE s.c. treatment phase (including PE tapering if            applicable, and study drug post-PE for 30 days after the            very last PE)    -   Follow-up phase

The patients received the best medical care and treatment judgedappropriate by the investigator at each site and according to theguidelines for treatment of TTP. The maximum total duration ofindividual study participation was a maximum of 15 months: a treatmentphase of up to 90 days and a follow-up period of maximum of 1 year afterremission or after 90 days of treatment, whichever came first. Ingeneral, patients were hospitalised for at least 1 day after the lastdaily PE.

The study drug was administered as an adjunctive treatment at specifictimes relative to PE procedures. The study drug consisted of 10 mg ofCaplacizumab (“treatment group”) or placebo (“placebo group”), once ortwice daily.

7.4.1 First Drug Administration

Patients received a first i.v. bolus of 10 mg ALX 0081 or placebo viapush injection 15 minutes to 6 h prior to the initiation of the firstPE. This first PE was followed by s.c. administration of 10 mg studydrug within 30 minutes after the end of the PE procedure.

During the complete PE treatment period (including tapering and PE givenfor exacerbations), the study drug was administered daily via s.c.injections.

If 1 PE per day was scheduled, 10 mg of study drug was administeredwithin 30 minutes after the end of the PE procedure.

If 2 PEs per day were scheduled, 10 mg of study drug was administeredwithin 30 minutes after the end of each PE procedure. The maximum totaldaily dose of study drug was hence 20 mg.

If less than 1 PE per day was scheduled (i.e. during a taperingregimen), 10 mg of study drug was administered daily. On days with a PE,study drug administration was within 30 minutes after the end of the PEprocedure; on days without PE, study drug administration was 24 h (±1 h)after previous administration.

Daily s.c. study drug administration of 10 mg continued for 30 daysafter the very last PE (including tapering).

7.4.2 Primary Endpoint

The primary endpoint of this phase II study was the time-to-response(TTR), based on the following criterion: recovery of platelets≥150,000/μL. In order to qualify as meeting the endpoint, the responsehad to be confirmed at 48 hours after the initial reporting of plateletrecovery equal to or above 150,000/μL by a de novo measure of platelets≥150,000/μL and lactate dehydrogenase (LDH) ≤2× upper limit of normal(ULN), i.e., “confirmed platelet response”. Platelet count is thepivotal laboratory marker for therapeutic decision making in patientswith TTP. This is based on the fact that ULvWF-mediated plateletaggregation is the common pathophysiological mechanism behind TTP,leading to severe thrombocytopenia and microangiopathic hemolyticanemia, which are the main hallmarks in the diagnosis of TTP (Scully etal. supra).

7.4.3 Determination of ADAMTS13 Activity

ADAMTS13 activity and functional inhibitor activity were measured by afluorogenic assay using the FRETS-VWF73 substrate (Kokame et al. 2005.Br J Haematol 129(1):93-100; Kremer Hovinga et al. 2006 J Thromb Haemost4(5):1146-8),

Briefly, the FRETS-VWF73 assay were performed essentially as described(Kokame et al. 2005 supra) with the following modifications: Pefabloc SC(Boehringer, Mannheim, Germany) was added to the assay buffer (5 mmolL-1 Bis-Tris, 25 mmol L-1 CaCl2, 0.005% Tween-20, pH 6.0) at a finalconcentration of 1 mmol L-1. Assay calibration was obtained by using anormal human plasma pool (NHP; Swiss Red Cross Blood Services, Bern,Switzerland) diluted 1:25 (100%) in assay buffer. Further calibrationsamples were obtained by serial predilutions of NHP of 3:4 (75%), 1:2(50%), 1:4 (25%), 1:10 (10%), 1:20 (5%), 1:50 (2%) and 1:100 (1%) inheat-inactivated NHP, incubated for 30 min at 56° C. followed by 15 minof centrifugation at 15 000×g) to correct for a plasma matrix effect inthe lower activity range of the standard curve. All of these standardsamples as well as heat-inactivated NHP (0% ADAMTS13 activity) and alltest samples were subsequently diluted 1:25 in assay buffer. Next, 25 μLof each diluted standard or patient sample was incubated at 37° C. in a384-well white plate (NUNC, Roskilde, Denmark). After 10 min, 25 μl of 4μmol L-1 FRETS-VWF73 peptide substrate dissolved in assay buffer wasadded to each well and evolution of fluorescence recorded at 37° C. in afluorescence microplate reader (GENios, Tecan, Zurich, Switzerland)equipped with a 340 nm excitation filter (band width 35 nm) and a 450 nmemission filter (band width 25 nm). Fluorescence evolution was measuredover time (every 5 min for 42 cycles). The reaction rate was calculatedby linear regression analysis (Passing-Bablok) of fluorescence evolutionover time from 5 (cycle 2) to 60 min (cycle 13). The slope of theregression curve was calculated for each calibration sample, and used togenerate the calibration curve (trend line: y=ax+b; with x=ADAMTS13(%)and y=delta RFU/delta time). The ADAMTS13 activity (%) of a sample wasthen calculated as: (y−b)×1/a.

ADAMTS13 functional inhibitor activity was measured by the samefluorogenic FRETS-VWF73 method by determination of residual ADAMTS13activity of normal human plasma after 1:1 (v:v) incubation for 2 hoursat 37° C. with heat-inactivated patient's plasma (30 min at 56° C.).

For each analytical batch, a calibration curve was generated using anormal human plasma pool (NHP; Swiss Red Cross Blood Services, Bern,Switzerland) diluted 1:25 (100%) in assay buffer. Further calibrationsamples were obtained by serial predilutions of NHP of 1:2 (50%), 1:4(25%), 1:10 (10%), 1:20 (5%), 1:50 (2%) and 1:100 (1%) inheat-inactivated NHP. All calibration points were applied in singlicate.Acceptance criteria: (1) The slope of the final regression of thestandard curve line has to be >6.0; and (2) R² of the regression of thefinal plot has to be >0.98 (or R >0.9899). Otherwise, the assay wasrejected.

7.5 Results in TTP Patients 7.5.1 Subject Disposition and Analysis ofPopulations.

The phase II study comprised a sample size of 75 patients, which wererandomized as set out in Table 2.

The primary analysis population was the intent-to-treat (ITT)population, which consisted of all randomised subjects according to arandomised treatment assignment. In addition, for the efficacy analyses,the per protocol population (PP) was used. The PP population is a subsetof the ITT population and consists of all randomised subjects, accordingto the randomised treatment assignment, with exclusion of all majorprotocol deviations and violators.

TABLE 2 Caplacizumab Placebo Total N (%) N (%) N (%) Randomized 36 39 75Not treated 1 (2.8%) 2 (5.1%) 3 (4.0%) ITT population 36 (100%)  39(100%)  75 (100%)  Safety population 35 (97.2%) 37 (94.9%) 72 (96.0%)

In conclusion, there was an even distribution in both treatment arms,i.e. patients receiving Caplacizumab (treatment group) and patientsreceiving placebo (placebo-group). The treatment arms were furthermorewell-balanced for age, ethnicity/race and BMI.

The base line characteristics of various parameters were assessed in thepatients of the treatment group and the placebo group. In Table 3 thebaseline platelet counts and LDH are presented. The increased of LDHlevels is a sign of increased haemolysis and/or tissue ischaemia.

TABLE 3 Caplacizumab Placebo Total N = 35 N = 37 N = 72 Platelets(10³/mm³) mean ± St Dev 21.1 ± 18.2 28.0 ± 20.0 24.6 ± 19.3 minimum,maximum 2, 70 5, 84 2, 84 LDH (U/L) mean ± St Dev 1277.4 ± 852.5  1270.1± 939.3  1273.7 ± 891.0  minimum, maximum 240, 3874 247, 4703 240, 4703

There is a slightly lower mean baseline platelet count for theCaplacizumab arm. In both arms, the mean platelet counts were very lowat baseline, which is consistent with a severe disease setting but isalso indicative that all subjects presenting were considered forinclusion and there is no bias towards less severely thrombocytopenicsubjects.

LDH means are comparable for both treatment arms.

In Table 4 the baseline vWF:Ag and ADAMTS13 activity are presented.

TABLE 4 Caplacizumab Placebo Total N = 36 N = 39 N = 75 vWF:Ag (%) mean± St 180.3 ± 78.2 185.5 ± 80.8 183.1 ± 78.9 Dev; ULN = 150 ADAMTS13activity n (%) <5%, indicative of 21 (58.3%)  22 (56.4%) 43 (57.3%)idiopathic TTP ≥5% 9 (25.0%) 14 (35.9%) 23 (30.7%) missing 6 (16.7%) 3(7.7%) 9 (12%) 

Both treatment arms were well balanced for vWF:AG and ADAMTS13 activity.More than half of the subjects have idiopathic TTP as indicated by <5%ADAMTS13 activity.

7.5.2 Study Results: Primary Endpoints

The time-to-response of blood markers was monitored in a survivalsetting. The primary endpoint time-to-response of blood markerscomprised recovery of platelets >150,000/μL. The platelet levelsrepresent a reliable surrogate marker for TTP disease activity. Zero totime-to-event period was set at 30 days.

The results are depicted in Table 5.

Data were evaluated according to stratification (1 PEX prior toRandomization: YES & NO), which aggregates to an overall Hazard Ratiofor the complete ITT population of 2.197 with a p value=0.013 for theoverall ITT population. The Hazard Ratio means that at any time,subjects receiving Caplacizumab have more than twice the rate ofachieving the primary endpoint of confirmed platelet recovery incomparison to subjects on Placebo.

Reduction in time to Confirmed Platelet Response (primary endpoint):

-   -   In the group of subjects with no PEX prior to randomization, a        median of 4.92 days for the Placebo arm reduced to 3.00 days for        the Caplacizumab arm: 39% reduction (PEX prior to        Randomization=NO)    -   In the group of subjects which received one PEX prior to        randomization, a median of 4.31 days for the Placebo arm reduced        to 2.44 days for the Caplacizumab arm: 43% reduction (PEX prior        to Randomization=YES)

The median times of 4.31 days and 4.92 days for the Placebo arms of the2 strata (1 PEX prior to Randomization: YES & NO) are lower than thatexpected from medical literature and investigators' data (6 days; cf.Bandarenko et al. Journal of Clinical Apheresis 1998; 13: 133-141). Thisimplies that even with an improved standard of care treatment overhistorical data, the Caplacizumab treatment was superior.

The 95% CI of the time to Confirmed Platelet Response is 2 to 3 timesnarrower for the Caplacizumab arms versus the Placebo arms. This impliesthat the time to disease resolution is less variable in the Caplacizumabtreatment arms than the Placebo arms,

TABLE 5 PEX Caplacizumab Placebo prior * N = 36 N = 39 No Subjectscensored at 30 days n (%) 5 (13.9%) 11 (28.2%) No Subjects withconfirmed platelet 29 (80.6%) 24 (61.5%) response n (%) No Median (95%CI) 3.00 (2.74, 3.88) 4.92 (3.21, 6.59) No 25^(th) & 75^(th) percentile2.72 & 4.31 3.01 & 11.37 Yes Subjects censored at 30 days n (%) 0 0 YesSubjects with confirmed platelet 2 (5.6%) 4 (10.3%) response n (%) YesMedian (95% CI) 2.44 (1.92, 2.97) 4.31 (2.91, 5.68) Yes 25^(th) &75^(th) percentile 1.92 & 2.97 3.37 & 5.23  Overall Hazard Rate Ratiofor Caplacizumab 2.197 (1.278, 3.778) versus Placebo (95% CI) StratifiedLog-rank Test p-value 0.013 * 1 PEX prior to randomization

7.53 Study Results: Exacerbations

Within the ITT population, the proportion of subjects with exacerbationswas determined. Exacerbation refers to a recurrent thrombocytopeniafollowing a confirmed platelet response and requiring a re-initiation ofdaily PE treatment after ≥1 day but ≤30 days after the last PE.

The results are depicted in Table 6.

TABLE 6 Caplacizumab Placebo Total N = 36 N = 39 N = 75 OverallPopulation 3 (8.3%) 11 (28.2%) 14 (18.7%)

There are 3× more exacerbations in the Placebo arm versus theCaplacizumab arm.

This confirms the finding that the polypeptide of the invention, such asALX 0081, can be solely responsible for treating and/or alleviating (thesymptoms of) TTP.

7.5.4 Study Results: Relapses

The number of subjects relapsing of TTP was assessed. Relapse of TTP isdefined as a de novo event of TTP that occurs later than 30 days afterthe last daily PE. In Table 7 the proportion of subjects withexacerbation and/or relapse in the 1st month after end of treatment inthe ITT Population is depicted.

TABLE 7 Caplacizumab Placebo Total N = 36 N = 39 N = 75 OverallPopulation 13 (36.1%) 13 (33.3%)  26 (34.7%) Relapse (at least) 10 2 12Baseline ADAMTS13 activity n (%) <5%  7 (19.4%) 8 (20.5%) 15 (20.0%) ≥5%2 (5.6%) 5 (12.8%) 7 (9.3%)

We see more Relapses in the Caplacizumab arm versus the Placebo (PLC)arm, which evens out the total numbers.

The Relapses in the Caplacizumab arm occur often within a few days ofthe Caplacizumab treatment termination. This implies a possible‘extended’ exacerbation rather than a relapse. Therefore Caplacizumab(CAP) treatment may possibly have been too short duration for somesubjects. Indeed, higher number of early relapses in the CAP armsubstantiates a protective effect and warrants longer CAP treatment insome patients. This implies that Caplacizumab treatment should becontinued for prolonged periods.

It is interesting to note that in both treatment arms, relapses are moreprominent in patients with a baseline ADAMTS13 activity of <5%, eventhough the ADAMTS13 activity was only available in a subset of thepatients. This may indicate that patients with baseline ADAMTS13activity of <5% are more prone to relapses (or exacerbations) andCaplacizumab treatment should be continued for even longer periodscompared to patients with higher activity (cf. Example 7.5.8).

7.5.5 Study Results: Complete Remission

The number of subjects with complete remission in the ITT population wasassessed. Complete remission is here defined as confirmed plateletresponse and the absence of exacerbation. The results are depicted inTable 8.

TABLE 8 Caplacizumab Placebo N = 36 N = 39 Overall Number of subjects n(%) 29 (80.6%) 18 (46.2%) 95% CI (64.0%, 91.8%) (30.1%, 62.8%) BaselineADAMTS13 Number of subjects n (%) 17 (47.2%) 12 (30.8%) activity <5% 95%CI (58.1%, 94.6%) (32.2%, 75.6%) Baseline ADAMTS13 Number of subjects n(%)  7 (19.4%)  6 (15.4%) activity ≥5% 95% CI (40.0%, 97.2%) (17.7%,71.1%)

The Caplacizumab arm presents 1.6× more subjects with complete remissionversus the Placebo arm. The greater proportion of complete remission,coupled with the faster and narrower distribution of time to confirmedplatelet response supports a greater predictability of patient responseto PE in the CAP arm, and is reflected in number of days of PE. Thenumber of consecutive days of PE was (mean±st dev) 6.6±3.4 days for CAPversus 8.1±6.5 days for PLC with a total plasma volume administeredincluding tapering of 22.5±15.9 liters for CAP versus 28.4±21.3 litersfor PLC. The effect appears more pronounced for the subgroup of subjectswith low Baseline ADAMTS13 activity, even though the baseline ADAMTS13activity data is incomplete for the Caplacizumab group (cf. Example7.5.8).

7.5.6 Study Results: Safety Assessment

The incidence of adverse events (AE) and serious adverse events (SAE)was assessed, focusing on bleeding related SAEs and immune-related SAEs.The SAE assessment included haemorrhage from catheter insertion, sepsis,catheter thrombosis, pneumothorax, fluid overload, hypoxia, hypotension,anaphylactoid reactions and TRALI. The number of serious adverse eventswas similar across both treatment arms: 57% in the Caplacizumab armcompared to 51% in the placebo arm. The number of adverse events wasalso similar across treatment arms: 97% in the Caplacizumab arm comparedto 100% in the placebo arm. The number of subjects with bleeding relatedAEs was slightly elevated in the Caplacizumab arm (54%) compared to theplacebo arm (38%),

The most prominent risk of the currently used non-specificantithrombotic agents is an elevated bleeding diathesis or apparentbleeding. Beside any unexpected effects, bleeding also represents themost relevant safety concern for Caplacizumab. In this contextCaplacizumab was investigated in a preclinical surgical bleeding model.In this study, surgical blood loss in animals receiving Caplacizumab wascomparable to blood loss in Heparin® treated animals, and 2- and 4 foldless than in Plavix® and ReoPro® treated animals, respectively. Althoughthis is a positive indication that Caplacizumab may be safer thanPlavix® and ReoPro® in terms of bleeding risk, this was assessed inhealthy persons. As indicated above, TTP-patient differs in many aspectsfrom a healthy person in respect of vWF. Study treatment was stopped dueto an adverse event in 4 patients treated with Caplacizumab and in 2patients treated with placebo. The increase in bleeding tendency washowever well manageable.

TTP is potentially life threatening. There were 2 deaths (5.4%) reportedin the trial, both in the placebo arm. It is noted that the number ofdeaths in the placebo arm is less than described in literature (10-30%).This may infer that the outcome of TTP has improved due to moreeffective standards of care (in the present study).

7.5.7 Study Results: Further Optimized Treatment Protocol

A further optimized treatment protocol was designed by the presentinventors, based on the idea that the distribution of confirmed plateletresponse time is shorter and not skewed and biased to the right (longertime to response) in the CAP arm in comparison to the placebo arm.

In the further optimized treatment protocol, all subjects are treated inessence as set out before in point 7.4, “Study Design”, but with thefollowing major modification: the PE treatment period is set for 3-5days, such as 3 days or 4 days or 5 days, preferably 3 days. The PEtreatment period is independent of the recovery of platelets(≥150,000/μl). Daily s.c. study drug administration is continued for atleast 10 days, such as 20 days or 30 days after the very last PE, butpreferably for at least 10 days, such as 20 days or 30 days after therecovery of platelets to ≥150,000/μl. In evaluating the furtheroptimized treatment protocol, the primary endpoint will be the number ofexacerbations as defined supra.

In the further optimized treatment protocol, the burden for the patientand the costs are decreased.

7.5.8 ADAMTS13 Activity

It was set out to further evaluate ADAMTS13 activity as marker forunderlying disease activity and to evaluate ADAMTS13 activity as markerto guide optimal treatment duration of caplacizumab, to maintaintreatment benefit.

In this case, when ADAMTS13 activity was <10% then underlying diseaseactivity was assumed. An ADAMTS13 activity of ≥10% assumed no underlyingdisease activity.

Exacerbations and relapses were related to the patient's availableADAMTS13 activity data. If a relapse was preceded by continuously lowADAMTS13 (<10%) during treatment, it was considered as relapse ofpresenting episode. If a relapse was preceded by ADAMTS13 activity ≥10%during treatment, it was considered as a de novo TTP episode. Patientsexcluded from analysis were (i) non-ADAMTS13 mediated disease (≥10% atbaseline) (n=3), and (ii) patients with insufficient data (n=12).

(i) Patients without Exacerbations or Relapses:

-   -   Caplacizumab: 13/16 (81%) had ADAMST13 activity values ≥10%        close to treatment stop    -   Placebo: 14/16 (88%) had ADAMST13 activity values ≥10% close to        treatment stop

Hence, in the majority of patients without exacerbation or relapse,ADAMTS13 activity recovered to levels above 10%, suggesting resolutionof the presenting TTP episode.

(ii) Patients with Exacerbations

-   -   Caplacizumab: ⅔ (67%) had ADAMTS13 activity <10% around their        exacerbation    -   Placebo: ⅞ (88%) had ADAMTS13 activity <10% around their        exacerbation

Hence, in the majority of patients with exacerbations of TTP, ADAMTS13activity was below 10%, suggesting unresolved disease activity leadingto exacerbation.

(iii) Patients with Relapses of the Presenting TTP Episode

-   -   Caplacizumab: 7/7 (100%) had ADAMST13 activity values <10% close        to treatment stop; relapses occurred within 10 days after        treatment stop    -   Placebo: N/A—all were ‘de novo’ relapses

Hence, all subjects in the caplacizumab group with relapses occurringsoon after the end of treatment had continuously low ADAMTS13 activity(<10%), indicative of ongoing disease.

(iv) Patients with a De Novo Relapse Episode

-   -   Caplacizumab: 4/4 (100%) had ADAMST13 activity values ≥10% close        to treatment stop; relapses occurred within ≥30 days after        treatment stop (range: 30-167 days). All 4 patients had again        ADAMTS13 activity <10% at the time of relapse.    -   Placebo: ⅔ (67%) had ADAMST13 activity values ≥10% close to        treatment stop; relapses occurred within ≥30 days after        treatment stop (range: 30-167 days). There were no data        available around the time of relapse.

Hence, in the majority of patients with a de novo relapse episode,ADAMTS13 activity recovered to levels above 10% near treatment stop,suggesting resolution of the presenting TTP episode; values were lowagain (<10%) at the time of relapse, indicating a new TTP episode

(v) Conclusion

The data support the use of ADAMTS13 activity as predictive marker forrecurrences of TTP and its potential for treatment decisions.

ADAMTS13 activity is able to predict relapses which occur shortly afterstopping caplacizumab treatment.

These relapses are considered as relapses of the presenting TTP episode(unresolved disease activity, based on continuously low ADAMTS13activity).

A 30-day treatment period (post PE) with caplacizumab has demonstrated asignificant impact on the number of exacerbations.

Extending the caplacizumab treatment period for those patients at riskfor relapse (i.e. with underlying disease activity based on ADAMTS13activity) will maintain the protective effects of caplacizumab until theunderlying disease is adequately treated and resolved.

Conversely, precautionary treatment with caplacizumab will reduce therisk of an acute episode of TTP.

7.5.9 Study Results: Organ Damage Markers

The characteristic microvascular occlusions in TTP patients can lead toorgan ischaemia throughout the body including brain, heart and, to alesser extent, kidneys.

Acute myocardial infarction (AMI) has been reported as an earlycomplication of TTP based both on clinical diagnosis of AMI (Patschan etal. 2006 Nephrology, dialysis, transplantation 21: p. 1549-54) andautopsy findings (Hosier et al. Archives of pathology & laboratorymedicine, 2003. 127(7): p. 834-9). Based on high serum lactatedehydrogenase (LDH) and troponin I (TnI) elevations it was demonstratedthat patients with clinically suspected TTP are at high risk to developAMI. Raised troponin T levels were linked with mortality and acutemorbidity. Subjects that died had higher troponin T levels, while therewere no deaths in the group of normal troponin T levels. Histologyconfirmed widespread myocardial microvascular thrombosis in the subjectsthat died (Hughes et al. 2009 J. Thromb. Haemost. 7: p. 529-536). Theseresults suggest that more rapidly reducing further microvascular cardiacischemia, e.g. measured by troponin, of the presenting TTP episode couldbe expected to have a benefit on clinical outcome, e.g. reduces the riskon organ damage, such as brain, heart and kidneys. Other retrospectivereports found similar results on acute and long-term cardiac involvementin TTP patients including myocardial infarctions (Wahla et al. 2008 Eur.J. Haem. 81: p. 311-6) and other cardiac events like infarctions,arrhythmias, cardiogenic shock and sudden cardiac death (Hawkins et al.2008 Transfusion 48: p. 382-92).

In a retrospective study, high levels of LDH at presentation were linkedwith a worse long term outcome (mortality), reflective of a severemultiple organ involvement (Benhamou, et al. 2012 Haematologica 97: p.1181-6). Hence, a fast normalization of LDH as marker for hemolysis andischemic organ damage may also be beneficial for long term clinicaloutcome, and reduces the risk on organ damage.

Since ischaemic damage may result in both acute complications as well asin poorer longer term outcomes, an analysis was performed on specificand clinically relevant organ damage biomarkers of LDH, troponin T or Iand creatinine. It was considered that cardiac troponin (I or T) is aspecific marker for myocardial damage, creatinine is a biomarker forrenal function, and LDH is a marker of haemolysis and even predominantlya marker of organ damage in this disease. In TTP patients, high baselinelevels or a slower normalisation in some of these biomarkers have beenlinked with worse clinical outcomes (e.g. mortality, refractorydisease).

The data suggest that more rapidly curtailing microvascular tissueischemia as measured by organ damage biomarkers is expected to have abenefit on clinical outcome, e.g. a reduced risk on organ damage.However, it should be noted that the organ damage marker results are tosome extent confounded by the dilutive effect of daily plasma exchange(both in the patient and placebo group).

(i) Lactate Dehydrogenase

LDH is an important marker of non-specific tissue ischemia. Proportionswere calculated based on the number of subjects in the ITT Population.The number and proportion of subjects in the ITT Population with valuesfor LDH ≤2×ULN was summarized by planned treatment for the first 5 studydays. A summary of results is provided in Table 9. The mean LDH/ULNratio by study day is provided in Table 10.

On Day 1, 11 subjects (30.6%) in the ALX-0081 treatment group and 9subjects (23.1%) in the placebo group had LDH values ≤2×ULN. Theproportion of subjects remained higher in the ALX-0081 treatment groupcompared with the placebo group on Days 2 (28 subjects [77.8%] and 20subjects [51.3%], respectively) and 3 (33 subjects [91.7%] and 29subjects [74.4%], respectively). On Days 4 and 5, the proportion ofsubjects with values for LDH ≤2×ULN was similar in both groups.

Mean ratios of LDH/ULN were comparable between the ALX 0081 and placebotreatment groups on Day 1 (3.93 and 3.98, respectively) and higher inthe placebo group at all other time points included.

TABLE 9 Number and Proportion of Subjects in with Lactate DehydrogenaseValues ≤2 × the Upper Limit of Normal by Study Day (Intent-To-TreatPopulation) Placebo ALX 0081 N = 36 N = 39 Analysis Relative Day n (%) n(%) 1 11 (30.6)  9 (23.1) 2 28 (77.8) 20 (51.3) 3 33 (91.7) 29 (74.4) 434 (94.4) 34 (87.2) 5 34 (94.4) 34 (87.2) N = number of subjects in thepopulation of interest; n = number of subjects with LDH ≤2 × the ULN;ULN = upper limit of the normal range

TABLE 10 Mean Lactate Dehydrogenase/Upper Limit of Normal Ratio by StudyDay (Intent-To-Treat Population) ALX 0081 Placebo Analysis Day RelativeN = 36 N = 39 1 n 34 35 Mean 3.93 3.98 2 n 35 36 Mean 1.70 2.03 3 n 3536 Mean 1.05 1.44 4 n 35 36 Mean 0.90 1.25 5 n 35 36 Mean 0.88 1.14 N =number of subjects in the population of interest; n = number of subjectswith available data

A Kaplan-Meier analysis was conducted to compare the time tonormalization of LDH values according to the planned treatment. Time toLDH normalization curves are provided in FIG. 2.

From the Kaplan-Meier analysis a more rapid return to normal levels ofLDH is suggested for subjects receiving ALX 0081 as compared to subjectsreceiving placebo.

(ii) Troponin

A Kaplan-Meier analysis was conducted to compare the time tonormalization of Troponin T or Troponin I values according to theplanned treatment. As noted above, both TnT and TnI are relevantbiomarkers of cardiac cell damage. Time to Troponin T or I normalizationcurves are provided in FIG. 3.

From the Kaplan-Meier analysis a more rapid return to normal levels ofTroponin T or I is suggested for subjects receiving ALX 0081 as comparedto subjects receiving placebo.

(iii) Creatinine

Also in this case a Kaplan-Meier analysis was conducted to compare thetime to normalization of creatinine values according to the plannedtreatment. Time to creatinine normalization curves are provided in FIG.4.

For creatinine a more rapid return to normal levels for subjectsreceiving ALX 0081 as compared to subjects receiving placebo issuggested by the Kaplan-Meier analysis.

(iv) Discussion

Given the pathophysiology of acquired TTP whereby ULvWF strings consumeplatelets in the formation of microthrombi, it was reasoned that therecovery of platelet counts is an indirect measure of prevention offurther microthrombi formation. The morbidity and the acute mortalityassociated with acquired TTP is a result of these microthrombi.

Indeed, this reasoning is supported by the organ damage markers. Inparticular, the results indicate that the organ damage markers troponinI and T, LDH and creatinine return faster to normal levels in subjectsreceiving ALX 0081 than in subjects receiving placebo.

Hence, the results suggest that a faster normalization rate of theseorgan damage markers is linked to a better clinical outcome, i.e. areduced risk of and less organ damage due to organ ischemia caused bymicrothrombi.

7.5.10 Dose Selection Rationale

The pharmacology of caplacizumab is two-fold. Caplacizumab affects thefunctionality of vWF, leading to inability of vWF to bind to platelets.It also affects the disposition of vWF, leading to transient reductionsof total vWF:Ag levels during treatment.

A PK/PD model was used to evaluate the expected exposure andcorresponding effect on total, free and complexed vWF levels fordifferent dosing scenarios in a virtual TTP patient population, throughsimulations. A virtual population of TTP patients (n=500), withcharacteristics based on the population from the TITAN study, wascreated by sampling the truncated distributions of body weights (mean81.9 kg±22.6 SD, range 47.5-150 kg) and model-estimated baseline vWF inthe TTP population of the TITAN trial as well as gender distribution(M:F 40:60).

Simulated scenarios included an initial 8 days daily PE procedure with aconcomitant s.c. daily administration of caplacizumab 1 h after thetermination of each PE at doses of 2.5, 5, 10, 20 mg (period 1).Subsequently, caplacizumab was assumed to be administered at the samedose levels/regimen for additional 30 days in absence of any other PEprocedure (period 2).

The % change of the free vWF from baseline increases with the dose, butless than dose-proportional. A large inter-individual variability,mainly related to the large variability of target expression (vWF) atstudy entry, is predicted at all the dose levels. The two lower doses(2.5 and 5 mg once daily) lead to a sub-optimal target inhibition,whereas a higher daily dose of caplacizumab than the one tested in theTITAN study (20 mg) does not substantially benefit the overall simulatedTTP population. FIG. 5 shows the model-predicted % decrease frombaseline of free vWF:Ag levels at the end of period 2 as a function ofthe dose level, including patients treated with placebo.

The simulated plasma profiles of the drug, free, complex and total vWFlevels for a 10 mg once daily dose are illustrated in FIG. 6.

Next to change in levels of the free vWF as marker for targetneutralization, the change in levels of total vWF as marker for bleedingrisk was evaluated. A threshold of 0.4 IU/mL (or 16 nM) was used, linkedto the highest vWF levels observed in von Willebrand's disease type I.It is considered that at these levels of vWF, sufficient levels of FVIIIremain available. Panel D shows that total vWF levels for the proposeddose of 10 mg once daily would remain above this threshold consideredfor risk of bleeding events.

Based on the previous in vitro study results and model-predicted levelsof biomarkers for efficacy (free vWF) and safety (total vWF), the dosingregimen used herein (5-40 mg, preferably 10 mg daily) is considered asadequate for reaching the desired suppression of the platelet-bindingcapacity of (UL)vWF in TTP patients, while bleeding events are minimaland at least controllable.

7.6 Conclusion

There is a large inter-individual variability of target expression (vWF)at study entry. Nevertheless, the doses used lead to an optimal targetinhibition, substantially benefitting the overall treated TTPpopulation.

Hence, ALX 0081 represents a novel approach to the treatment of TTP andprovides a significant benefit in terms of efficacy, safety and qualityof life for patients with TTP.

Proof of concept of Caplacizumab was demonstrated with statisticallysignificant and clinically meaningful reduction of time to confirmedplatelet response. The median days to confirmed platelet response was 3days for Caplacizumab vs. 4.9 days for Placebo. The HR (Placebo overCaplacizumab) of 2.2 with 95% CI (1.28, 3.78), p=0.013.

There was a reduction in the number of exacerbations to 3 in theCaplacizumab arm from 11 in the Placebo arm. This underscores theprotective effect of caplacizumab treatment.

There were no deaths in the Caplacizumab arm compared to 2 deaths in thePlacebo arm.

The AEs and SAEs are consistent with serious, potentiallylife-threatening condition. Nearly twice as many bleeding events inCaplacizumab arm (66 events) vs. Placebo arm (35 events), although only5 SAEs in 2 subjects for Caplacizumab arm vs. 2 SAEs in 2 subjects forPlacebo arm.

Overall, the benefit risk assessment for patients with acquired TTP isvery positive.

Through its inhibition of ULvWF-mediated platelet aggregation andresulting antithrombotic effect the polypeptides of the invention, suchas ALX 0081, permits a more rapid control of acute bouts of TTP whenused in combination with PE and transfusion. This clearly reduces therisk of organ ischaemia. The more rapid normalisation of the plateletcount also reduces the risk of haemorrhagic complications. Its use alsoresults in improved outcomes in poorly responsive patients, includingthose with secondary TTP where mortality from the disease remains high.

In addition, the polypeptides of the invention, such as ALX 0081, are ofvalue in the prevention of relapses after recovery from an acuteepisode.

TABLE A-1 Examples of polypeptides comprising ISVDs and CDRs against vWFName SEQ ID NO Sequence 12A02H1-3a-12A02H1  1EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYY (ALX 0081)PDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02-3a-12A02  2QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02-GS9-12A02  3QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02-GS30-12A02  4QVKLEESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDLVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNNLKPEGTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A05-3a-12A05  5AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQG TQVTVSS12A05-GS9-12A05  6AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12A05-GS30-12A05  7AVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCLASGRIFSIGAMGMYRQAPGKQRELVATITSGGSTNYADPVKGRFTISRDGPKNTVYLQMNSLKPEDTAVYYCYANLKQGSYGYRFNDYWGQGTQVTVSS 12B06-3a-12B06  8QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSAAAEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12B06-GS9-12B06  9QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12B06-GS30-12B06 10QVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQAGGALRLSCAASGRTFSYNPMGWFRQAPGKERDVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNALKPEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H4-3a-12A02H4 11EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSAAAEVLQVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12B06H2-3a-12B06H2 12EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSAAAEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H1-GS9-12A02H1 13EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02H4-GS9-12A02H4 14EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12B06H2-GS9-12B06H2 15EVQVLESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H1-GS30-12A02H1 16EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMLSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12A02H4-GS30-12A02H4 17EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRSVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQGTQVTVSS 12B06H2-GS30-12B06H2 18EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKREVVAAISRTGGSTYYARSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYNFWGQGTQVTVSS 12A02H1 19EVQLVESGGGLVQPGGSLRLSCAASGRTFSYNPMGWFRQAPGKGRELVAAISRTGGSTYYPDSVEGRFTISRDNAKRMVYLQMNSLRAEDTAVYYCAAAGVRAEDGRVRTLPSEYTFWGQ GTQVTVSS12A02H1 CDR1 21 YNPMG 12A02H1 CDR2 22 AISRTGGSTYYPDSVEG 12A02H1 CDR3 23AGVRAEDGRVRTLPSEYTF

TABLE A-2 Name SEQ ID NO Sequence Human 20MIPARFAGVLLALALILPGTLCAEGTRGRSSTARCSLFGSDFVNTFDGSMYSFAGYCSYLLAG vWFGCQKRSFSIIGDFQNGKRVSLSVYLGEFFDIHLFVNGTVTQGDQRVSMPYASKGLYLETEAGYYKLSGEAYGFVARIDGSGNFQVLLSDRYFNKTCGLCGNFNIFAEDDFMTQEGTLTSDPYDFANSWALSSGEQWCERASPPSSSCNISSGEMQKGLWEQCQLLKSTSVFARCHPLVDPEPFVALCEKTLCECAGGLECACPALLEYARTCAQEGMVLYGWTDHSACSPVCPAGMEYRQCVSPCARTCQSLHINEMCQERCVDGCSCPEGQLLDEGLCVESTECPCVHSGKRYPPGTSLSRDCNTCICRNSQWICSNEECPGECLVTGQSHFKSFDNRYFTFSGICQYLLARDCQDHSFSIVIETVQCADDRDAVCTRSVTVRLPGLHNSLVKLKHGAGVAMDGQDIQLPLLKGDLRIQHTVTASVRLSYGEDLQMDWDGRGRLLVKLSPVYAGKTCGLCGNYNGNQGDDFLTPSGLAEPRVEDFGNAWKLHGDCQDLQKQHSDPCALNPRMTRFSEEACAVLTSPTFEACHRAVSPLPYLRNCRYDVCSCSDGRECLCGALASYAAACAGRGVRVAWREPGRCELNCPKGQVYLQCGTPCNLTCRSLSYPDEECNEACLEGCFCPPGLYMDERGDCVPKAQCPCYYDGEIFQPEDIFSDHHTMCYCEDGFMHCTMSGVPGSLLPDAVLSSPLSHRSKRSLSCRPPMVKLVCPADNLRAEGLECTKTCQNYDLECMSMGCVSGCLCPPGMVRHENRCVALERCPVFHQGKEYAPGETVKIGCNTCVCRDRKWNCTDHVCDATCSTIGMAHYLTFDGLKYLFPGECQYVLVQDYCGSNPGTFRILVGNKGCSHPSVKCKKRVTILVEGGEIELFDGEVNVKRPMKDETHFEVVESGRYIILLGKALSVVWDRHLSISVVLKQTYQEKVCGLCGNFDGIQNNDLTSSNLQVEEDPVDFGNSWKVSSQCADTRKVPLDSSPATCHNNIMKQTMVDSSCRILTSDVFQDCNKLVDPEPYLDVCIYDTCSCESIGDCACFCDTIAAYAHVCAQHGKVVTWRTATLCPQSCEERNLRENGYECEWRYNSCAPACQVTCQHPEPLACPVQCVEGCHAHCPPGKILDELLQTCVDPEDCPVCEVAGRRFASGKKVTLNPSDPEHCQICHCDVVNLTCEACQEPGGLVVPPTDAPVSPTTLYVEDISEPPLHDFYCSRLLDLVFLLDGSSRLSEAEFEVLKAFVVDMMERLRISQKWVRVAVVEYHDGSHAYIGLKDRKRPSELRRIASQVKYAGSQVASTSEVLKYTLFQIFSKIDRPEASRIALLLMASQEPQRMSRNFVRYVQGLKKKKVIVIPVGIGPHANLKQIRLIEKQAPENKAFVLSSVDELEQQRDEIVSYLCDLAPEAPPPTLPPHMAQVTVGPGLRNSMVLDVAFVLEGSDKIGEADFNRSKEFMEEVIQRMDVGQDSIHVTVLQYSYMVTVEYPFSEAQSKGDILQRVREIRYQGGNRTNTGLALRYLSDHSFLVSQGDREQAPNLVYMVTGNPASDEIKRLPGDIQVVPIGVGPNANVQELERIGWPNAPILIQDFETLPREAPDLVLQRCCSGEGLQIPTLSPAPDCSQPLDVILLLDGSSSFPASYFDEMKSFAKAFISKANIGPRLTQVSVLQYGSITTIDVPWNVVPEKAHLLSLVDVMQREGGPSQIGDALGFAVRYLTSEMHGARPGASKAVVILVTDVSVDSVDAAADAARSNRVTVFPIGIGDRYDAAQLRILAGPAGDSNVVKLQRIEDLPTMVTLGNSFLHKLCSGFVRICMDEDGNEKRPGDVWTLPDQCHTVTCQPDGQTLLKSHRVNCDRGLRPSCPNSQSPVKVEETCGCRWTCPCVCTGSSTRHIVTFDGQNFKLTGSCSYVLFQNKEQDLEVILHNGACSPGARQGCMKSIEVKHSALSVELHSDMEVTVNGRLVSVPYVGGNMEVNVYGAIMHEVRFNHLGHIFTFTPQNNEFQLQLSPKTFASKTYGLCGICDENGANDFMLRDGTVTTDWKTLVQEWTVQRPGQTCQPILEEQCLVPDSSHCQVLLLPLFAECHKVLAPATFYAICQQDSCHQEQVCEVIASYAHLCRTNGVCVDWRTPDFCAMSCPPSLVYNHCEHGCPRHCDGNVSSCGDHPSEGCFCPPDKVMLEGSCVPEEACTQCIGEDGVQHQFLEAWVPDHQPCQICTCLSGRKVNCTTQPCPTAKAPTCGLCEVARLRQNADQCCPEYECVCDPVSCDLPPVPHCERGLQPTLTNPGECRPNFTCACRKEECKRVSPPSCPPHRLPTLRKTQCCDEYECACNCVNSTVSCPLGYLASTATNDCGCTTTTCLPDKVCVHRSTIYPVGQFWEEGCDVCTCTDMEDAVMGLRVAQCSQKPCEDSCRSGFTYVLHEGECCGRCLPSACEVVTGSPRGDSQSSWKSVGQQWASPENPCLINECVRKEEVFIQQRNVSCPQLEVPVCPSGFQLSCKTSADDPSCRCERMEACMLNGTVIGPGKTVMIDVCTTCRCMVQVGVISGFKLECRKTTCNPCPLGYKEENNTGECCGRCLPTACTIQLRGGQIMTLKRDETLQDGCDTHFCKVNERGEYFWEKRVTGCPPFDEHKCLAEGGKIMKIPGTCCDTCEEPECNDITARLQYVKVGSCKSEVEVDIHYCQGKCASKAMYSIDINDVQDQCSCCSPTRTEPMQVALHCTNGSVVYHEVLNAMECKCSPRKCSK

1.-17. (canceled)
 18. A method of preventing (the symptoms of) anexacerbation of a vWF-related disease in a human, comprisingadministering to the human a dose of 1-80 mg, preferably 5-40 mg, evenmore preferably 10 mg of a polypeptide comprising two anti-human vonWillebrand Factor (vWF) immunoglobulin single variable domains (ISVDs).19. The method of preventing (the symptoms of) an exacerbation of avWF-related disease in a human according to claim 18, wherein saidpolypeptide comprises at least one ISVD binding SEQ ID NO:
 20. 20. Themethod of preventing (the symptoms of) an exacerbation of a vWF-relateddisease in a human according to claim 18, wherein at least one ISVD isrepresented by SEQ ID NO: 19 (12A02H1).
 21. The method of preventing(the symptoms of) an exacerbation of a vWF-related disease in a humanaccording to claim 18, wherein said polypeptide is at least 90%identical to SEQ ID NO:
 1. 22. The method of preventing (the symptomsof) an exacerbation of a vWF-related disease in a human according toclaim 18, wherein said polypeptide is ALX 0081 (SEQ ID NO: 1).
 23. Themethod of preventing (the symptoms of) an exacerbation of a vWF-relateddisease in a human according to claim 18, wherein said dose isadministered 1 time per day or two times per day.
 24. The method ofpreventing (the symptoms of) an exacerbation of a vWF-related disease ina human according to claim 18, wherein said step of administering thepolypeptide of the invention is repeated for at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 days, or even more than 10 days, such as 20 days,preferably more than 30 days, such as 2 months, 3 months, 4 months, 5months, 6 months or even more.
 25. The method of preventing (thesymptoms of) an exacerbation of a vWF-related disease in a humanaccording to claim 18, comprising repeating administering saidpolypeptide until the platelet number in said human is at least150,000/μl.
 26. The method of preventing (the symptoms of) anexacerbation of a vWF-related disease in a human according to claim 25,comprising repeating administering said polypeptide until the plateletnumber in said human is at least 150,000/μl on at least 2 consecutivemeasurements.
 27. The method of preventing (the symptoms of) anexacerbation of a vWF-related disease in a human according to claim 26,wherein said step of administering the polypeptide of the invention isrepeated for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or evenmore than 10 days, such as 20 days, preferably more than 30 days or evenmore, after said platelet number is at least 150,000/μl on at least 2consecutive measurements.
 28. The method of preventing (the symptoms of)an exacerbation of a vWF-related disease in a human according to claim26, wherein said 2 consecutive measurements are at least 24 h, morepreferably 48 h apart, such as at least 3 days apart, or even more suchas 4, 5, 6, or even 7 days apart, preferably a week apart.
 29. Themethod of preventing (the symptoms of) an exacerbation of a vWF-relateddisease in a human according to claim 18, wherein said vWF-relateddisease is chosen from acute coronary syndrome (ACS), transient cerebralischemic attack, unstable or stable angina pectoris, stroke, myocardialinfarction or thrombotic thrombocytopenic purpura (TTP), preferably TTP.30. A method of reducing the risk of exacerbations of a vWF-relateddisease in a human, by administering to the human a dose of 1-80 mg,preferably 5-40 mg, even more preferably 10 mg of a polypeptidecomprising two anti-human von Willebrand Factor (vWF) immunoglobulinsingle variable domains (ISVDs), wherein the risk of exacerbations isreduced by at least 10%, 20%, 30%, 40%, or even at least 50%, such as60%, 70%, 80%, 90% or even to 100%.
 31. The method of reducing the riskof exacerbations of a vWF-related disease in a human according to claim30, wherein said vWF-related disease is chosen from acute coronarysyndrome (ACS), transient cerebral ischemic attack, unstable or stableangina pectoris, stroke, myocardial infarction or thromboticthrombocytopenic purpura (TTP), preferably TTP.
 32. The method ofreducing the risk of exacerbations of a vWF-related disease in a humanaccording to claim 30, wherein said polypeptide is ALX 0081 (SEQ ID NO:1).
 33. The method of reducing the risk of exacerbations of avWF-related disease in a human according to claim 30, wherein said stepof administering the polypeptide of the invention is repeated for atleast 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or even more than 10 days,such as 20 days, preferably more than 30 days, such as 2 months, 3months, 4 months, 5 months, 6 months or even more.
 34. A method ofreducing the risk of exacerbations of a vWF-related disease in a human,by administering to the human a dose of 1-80 mg, preferably 5-40 mg,even more preferably 10 mg of a polypeptide comprising two anti-humanvon Willebrand Factor (vWF) immunoglobulin single variable domains(ISVDs), wherein the risk of exacerbations is reduced by a factor 2 ormore, such as 3, 4, 5, 6, 7, 8, 9, or even 10, or even more such as 20,50 or even
 100. 35. The method of reducing the risk of exacerbations ofa vWF-related disease in a human according to claim 34, wherein saidvWF-related disease is chosen from acute coronary syndrome (ACS),transient cerebral ischemic attack, unstable or stable angina pectoris,stroke, myocardial infarction or thrombotic thrombocytopenic purpura(TTP), preferably TTP.
 36. The method of reducing the risk ofexacerbations of a vWF-related disease in a human according to claim 34,wherein said polypeptide is ALX 0081 (SEQ ID NO: 1).
 37. The method ofreducing the risk of exacerbations of a vWF-related disease in a humanaccording to claim 34, wherein said step of administering thepolypeptide of the invention is repeated for at least 1, 2, 3, 4, 5, 6,7, 8, 9, or 10 days, or even more than 10 days, such as 20 days,preferably more than 30 days, such as 2 months, 3 months, 4 months, 5months, 6 months or even more.